Anti-inflammatory factor retentate, method of isolation, and use

ABSTRACT

The invention relates to an anti-inflammatory factor isolated from milk, methods of purifying the anti-inflammatory factor through ultrafiltration and diafiltration to produce Milk Protein Concentrate resulting in substantially or highly purified preparations and to methods for using this factor to remove adhered neutrophils from endothelial cells, to prevent the emigration of cells from the vasculature and to suppress the response of lymphocytes to foreign antigens.

CROSS-REFERENCE TO RELATED APPLICATION

This application is related to and claims benefit to U.S. ProvisionalPatent Application Ser. No. 62/098,988, filed Dec. 31, 2014, entitledANTI-INFLAMMATORY FACTOR RETENTATE, METHOD OF ISOLATION, AND USE, theentirety of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an anti-inflammatory factor, processesfor its production in substantially pure or highly pure form, and amethod for its use in the treatment of inflammation.

BACKGROUND OF THE INVENTION

Inflammation, as defined in Dorland's Medical Dictionary, is “alocalized protective response elicited by injury or destruction oftissues which serves to destroy, dilute or wall off both the injuriousagent and the injured tissue.” It is characterized by fenestration ofthe microvasculature, leakages of the elements of blood into theinterstitial spaces, and migration of leukocytes into the inflamedtissue. On a macroscopic level, this is usually accompanied by thefamiliar clinical signs of erythema, edema, tenderness (hyperalgesia),and pain. During this complex response, chemical mediators such ashistamine, 5-hydroxytryptamine, various chemotactic factors, bradykinin,leukotrienes, and prostaglandins are liberated locally. Phagocytic cellsmigrate into the area, and cellular lysosomal membranes may be ruptured,releasing lytic enzymes. All of these events may contribute to theinflammatory response.

Inflammation in patients with rheumatoid arthritis probably involves thecombination of an antigen (gamma globulin) with an antibody (rheumatoidfactor) and complement causing the local release of chemotactic factorsthat attract leukocytes. The leukocytes phagocytose the complexes ofantigen-antibody and complement and also release the many enzymescontained in their lysosomes. These lysosomal enzymes then cause injuryto cartilage and other tissues, and this furthers the degree ofinflammation. Cell-mediated immune reactions may also be involved.Prostaglandins are also released during this process.

Prostaglandins, which are likely to be generated in inflammation, causeerythema and increase local blood flow. Two important vascular effectsof prostaglandins are not generally shared by other mediators ofinflammation—a long-lasting vasodilator action and a capacity tocounteract the vasoconstrictor effects of substances such asnorepinephrine and angiotensin.

A number of mediators of inflammation increase vascular permeability(leakage) in the post-capillary and collecting venules. In addition,migration of leukocytes into an inflamed area is an important aspect ofthe inflammatory process.

The Arthus reaction is an inflammatory response brought about by theformation of immune complexes at subcutaneous sites where an antigencomplexes with antibody to that antigen. Neutrophils characteristicallyattach to the Fc portion of the immunoglobulin complex that forms at thesubcutaneous injection site where they release digestive enzymes,causing visible acute inflammation. Thus the reaction is primarilyneutrophil-mediated and agents that effect the development of thereaction do so via an effect on these cells.

There are several pathways whereby an agent might interfere withneutrophil migration from the blood vessels to an inflammatory site. Onelikely pathway is the inhibition of margination, the reversible“sticking” of inflammatory cells to the endothelial cell lining of theblood vessel wall. In the normal state about 50% of neutrophils arereversibly adhered, but during an acute inflammatory response, adhesionbecomes much stronger and is a key step in the process of neutrophilmigration. While prostaglandins are unlikely to be directly involved inthe chemotactic response, another product of the metabolism ofarachidonic acid, leukotriene, is a very potent chemotactic substance.

The anti-inflammatory response is any response characterized byinflammation as defined above. It is well known to those skilled in themedical arts that the inflammatory response causes much of the physicaldiscomfort, i.e., pain and loss of function, that has come to beassociated with different diseases and injuries. Accordingly, it is acommon medical practice to administer pharmacological agents which havethe effect of neutralizing the inflammatory response. Agents havingthese properties are classified as anti-inflammatory drugs.Anti-inflammatory drugs are used for the treatment of a wide spectrum ofdisorders, and the same drugs are often used to treat differentdiseases. Treatment with anti-inflammatory drugs is not for the disease,but most often for the symptom, i.e., inflammation.

The anti-inflammatory, analgesic, and anti-pyretic drugs are aheterogeneous group of compounds, often chemically unrelated, whichnevertheless share certain therapeutic actions and side-effects.Corticosteroids represent the most widely used class of compounds forthe treatment of the anti-inflammatory response. Proteolytic enzymesrepresent another class of compounds which are thought to haveanti-inflammatory effects. Hormones which directly or indirectly causethe adrenal cortex to produce and secrete steroids represent anotherclass of anti-inflammatory compounds. A number of non-hormonalanti-inflammatory agents have been described. Among these, the mostwidely used are the salicylates. Acetylsalicylic acid, or aspirin, isthe most widely prescribed analgesic-antipyretic and anti-inflammatoryagent. Examples of steroidal and non-steroidal anti-inflammatory agentsare listed in the Physician's Desk Reference, 1987 (see pp. 207 and 208for an index of such preparations).

The natural and synthetic corticosteroid preparations cause a number ofsevere side effects, including elevation of blood pressure, salt andwater retention, and increased potassium and calcium excretion.Moreover, corticosteroids may mask the signs of infection and enhancedissemination of infectious microorganisms. These hormones are notconsidered safe for use in pregnant women, and long-term corticosteroidtreatment has been associated with gastric hyperactivity and/or pepticulcers. Treatment with these compounds may also aggravate diabetesmellitus, requiring higher doses of insulin, and may produce psychoticdisorders. Hormonal anti-inflammatory agents which indirectly increasethe production of endogenous corticosteroids have the same potential foradverse side-effects.

The non-hormonal anti-inflammatory agents are synthetic biochemicalcompounds which can be toxic at high doses with a wide spectrum ofundesirable side-effects. For example, salicylates contribute to theserious acid-base balance disturbances that characterize poisoning bythis class of compounds. Salicylates stimulate respiration directly andindirectly. Toxic doses of salicylates cause central respiratoryparalysis as well as circulatory collapse secondary to vasomotordepression. The ingestion of salicylate may result in epigastricdistress, nausea, and vomiting. Salicylate-induced gastric bleeding iswell known. Salicylates can produce hepatic injury, and lead to aprolongation of clotting time. Therefore, aspirin should be avoided inpatients with severe hepatic damage, hypoprothrombinemia, vitamin Kdeficiency, or hemophilia, because the inhibition of platelet hemostasisby salicylates can result i n hemorrhage. Salicylate intoxication iscommon, and over 10,000 cases of serious salicylate intoxication areseen in the United States every year, some of them being fatal, and manyoccurring in children. See Goodman and Gilman's The PharmacologicalBasis of Therapeutics. 7th Ed., 1985. Accordingly, in spite of the largenumber of anti-inflammatory agents that are currently available, therestill exists a need for a safe, effective anti-inflammatory productwhich is free of side-effects and adverse reactions.

If a natural food product, such as one derived from milk, for example,could be obtained having anti-inflammatory effects, it would be aneasily administrable, readily available, safe therapeutic composition.

It has been known in the prior art to produce milks having a variety oftherapeutic effects. Beck, for example, has disclosed a milk containingantibody to Streptococcus mutans that has dental caries inhibitingeffect (U.S. Pat. No. 4,324,782). The milk is obtained by immunizing acow with S. mutans antigen in two stages and obtaining the therapeuticmilk therefrom.

Stolle et al. have disclosed a method for treating vascular disorders orpulmonary disorders associated with smoking in an animal which comprisesadministering to the animal milk collected from a cow being maintainedin a hyperimmune state (U.S. Pat. No. 4,636,384). Beck has disclosed amethod for treating inflammation in an animal which comprisesadministering to the animal an anti-inflammatory effective amount ofmilk collected from a cow maintained in an anti-inflammatory factorproducing state (U.S. Pat. No. 4,284,623). Heinbach, U.S. Pat. No.3,128,230, has described milk containing globulins of alpha, beta, andgamma components by inoculating a cow with antigenic mixtures. Petersonet al. U.S. Pat. No. 3,376,198), Holm (U.S. application (published) Ser.No. 628,987), Tunnah et al. (British Patent No. 1,211,87(⁻) and BiokemaS. A. (British Patent 1,442.283) have also described antibody-containingmilks.

None of the aforementioned references, however, disclose the identity ofthe component or components of therapeutic milks which produce thedesired therapeutic effects. For example, in Beck, U.S. Pat. Pat. No.4,284,623, the milk products used as a therapeutic means consist ofeither fluid whole milk, fluid fat-free whey, or whole milk powders.Although each of these milk products has anti-inflammatory properties,the factor or factors that actually provide the therapeutic benefitshave not yet been isolated or identified or purified to homogeneity.

A particular difficulty in obtaining highly purified preparations ofmilk anti-inflammatory factor(s) (MAIF) is the inability to removetightly bound salts from the MAIF by currently used purificationprocedures. One of the problems that this invention addresses, interalia, is the large scale preparation of MAIF including the eliminationof tightly bound salts from the MAT preparation, thereby resulting inhighly purified MAIF. Further, problems have previously arisen inobtaining highly pure, preparative scale preparations of MAIF whenbeginning with large volumes of starting materials (e.g. 90 liters ofskim milk). The invention provides solutions to these problems.

SUMMARY OF THE INVENTION

The present invention is directed to an anti-inflammatory factor presentin milk and various methods involving the use of the anti-inflammatoryfactor present in milk. Specifically, the invention is directed to ananti-inflammatory factor produced from milk by removing the fat from themilk; filtering the milk so as to remove molecules with molecularweights greater than about 10,000 daltons; fractionating the filtratecontaining small molecular weight molecules by ion-exchange; furtherenriching ion-exchange fractions in the factor by gel filtration andfurther enriching gel filtration fractions by affinity chromatographyusing a chromatography medium with an affinity for coplanar adjacent cishydroxyl groups.

The invention is further directed to methods for additional purificationof the milk anti-inflammatory factor using, inter alia, HPLC sizeexclusion chromatography and organic partition extraction protocols.

In an alternative embodiment, the present invention is directed to amethod for obtaining an anti-inflammatory factor from skimmed milkcomprising the steps of: (i) ultrafiltering the skimmed milk through afilter with a molecular weight cut-off of 1.000 daltons; (ii) collectingthe >1,000 dalton retentate from step (i), (iii) extracting theretentate from step (ii) by organic partition extraction and obtainingthe aqueous extract from the extraction; (iv) separating the aqueousextract from step (iii) by reversed-phase HPLC chromatography; and (v)collecting the eluate. This embodiment is particularly appropriate forlarge-scale preparation of milk anti-inflammatory factor in quantitiessuitable for handling, transportation, storage, and processing.

In a preferred embodiment, the skimmed milk is hyperimmune skimmed milk.

In a particularly preferred embodiment, the hyperimmune skimmed milk iswhey.

In another preferred embodiment, the organic partition extractionfurther comprises: (i) extracting with hexane and NH₄ OH; (ii)reextracting the aqueous phase from step (i) with ethyl acetate; and(iii) collecting said ethyl acetate extract.

In another preferred embodiment, the reversed-phase HPLC chromatographycolumn is a Zorbax SB-C₁₈ column.

In an alternative embodiment, the present invention is directed to ananti-inflammatory factor in highly purified form produced by a processcomprising: (i) ultrafiltering skimmed milk through a filter with amolecular weight cut-off of 1,000 daltons, (ii) collecting the >1,000dalton retentate from step (iii) extracting the retentate from step (ii)by organic partition extraction and obtaining the aqueous extract fromthe extraction; (iv) separating the aqueous extract from step (iii) byreversed phase HPLC chromatography; and (v) collecting the eluate.

In a preferred embodiment, the skimmed milk is hyperimmune skimmed milk.

In a particularly preferred embodiment, the hyperimmune skimmed milk iswhey and (MPC) Mild Protein Concentrate.

In another preferred embodiment, the organic partition extractionfurther comprises: (i) extracting with hexane and NH₄ OH; (ii)reextracting the aqueous phase from step (i) with ethyl acetate; and(iii) collecting said ethyl acetate extract.

In another preferred embodiment, the reversed-phase HPLC chromatographycolumn is a Zorbax SB-C₁₈ column.

It is understood that throughout this disclosure, wherever the term“skimmed milk” is used, the terms “milk” or “whole milk” may besubstituted.

The invention is further directed to methods for using a milkanti-inflammatory factor, including a milk anti-inflammatory factorproduced by any of the above alternative embodiments, to preventneutrophils from adhering to the endothelium of venules or to detachneutrophils which have already adhered to the endothelial cells liningthe walls of venules. In this way, the factor is used to reduce thetissue damage associated with the inflammatory response.

The invention is also directed to a method for using a milkanti-inflammatory factor, including the milk anti-inflammatory factor ofany of the above alternative embodiments to prevent interactions betweenCD18 cell-surface antigens and other molecules. It is known that suchinteractions are necessary for the exit of cells from the vasculatureand that such emigration leads to increased tissue damage in animalsduring the inflammatory response. CD18 antigens are also known to beimportant in the immunological response of a host organism to foreignantigens.

Also encompassed by the invention is the use of a anti-inflammatoryfactor, including the anti-inflammatory factor of any of the abovealternative embodiments, in mammals to prevent the emigration of cellsfrom the vasculature and to suppress the mitogenic response oflymphocytes to foreign antigens.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1. Isolation of the anti-inflammatory factor by ion-exchangechromatography on a column of DEAE-cellulose;

FIG. 2. Fractionation of the anti-inflammatory factor containing peak(second) from DEAE-cellulose chromatography (FIG. 1 on a Sephadex G-10molecular sieve column;

FIG. 3. Effect of immune milk on carrageenan-induced edema in rats (pawweight, % control paw, mean±sem, n=10);

FIG. 4. Effect of intraperitoneal administration of theanti-inflammatory factor on footpad edema in rats (μL, mean+SD, n=6);

FIG. 5. Intraperitoneal dose-response curve for the anti-inflammatoryfactor in rat paw edema test (% control, mea.n+SD, n=6);

FIG. 6. Effect of hyperimmune milk factor vs. placebo (lactose) onfootpad edema in rats (% control, mean±SD, n=6);

FIG. 7. Effect of iv and oral MAIF on footpad edema in rats (% control,mean+SD, n=6);

FIG. 8. Effect of low iv dosage of MAIF on footpad edema in rats (%control, mean+SD, n-6);

FIG. 9. Intravenous dose-response curve for MAIF in rat paw edema test(% control, mean±SD, n=6);

FIG. 10. Run 1, twin herd/ultrafiltration experiments (% average controledema, mean±SD, n=6);

FIG. 11. Run 2, twin herd/ultrafiltration experiments (% average controledema, mean±SD, n=6);

FIG. 12. Run 3, twin herd/ultrafiltration experiments (% average controledema mean±SD, n=6);

FIG. 13. Effect of various treatments of MAIF on inhibition of footpadedema in rats (μL footpad edema, mean±SD, n=6);

FIG. 14. Effect of fractions of MAIF and of immune wpc on inhibition offootpad edema in rats (μL footpad edema, mean±SD, n=6);

FIG. 15. Effect of five different anesthetics on the response tocarrageenan in the rat footpad. The accumulation of edema was monitoredat selected intervals in the same animals. n=6 for each data point;

FIG. 16. Demonstration a the biphasic nature of the response tocarrageenan in the rat footpad n=5 for each data point. Ether was usedas the anesthetic;

FIGS. 17A-B. MAIF, administered at either 5 mg per rat (A) or 40 mg perrat (B) does not inhibit the inflammatory response to carrageenan inether-anesthetized rats. n=4 for all data points;

FIG. 18. Suppression of carrageenan-induced edema accumulation duringthe secondary, phagocytic-cell mediated, response by 40 mg of MAIFinjected i.v. at the time of carrageenan challenge (time 0), n=12 foreach data point in the control group and n=10 for each data point in theMAIF-treated group;

FIG. 19. Effect of MAIF, given i.v. at 4 mg per rat at different times,on the response to carrageenan in the rat footpad. Edema was assessed 4hours after challenge in all cases. n=12 for each data point;

FIG. 20A-C. Effect of20 mg of MAIF injected i.v. on the reverse passiveArthus reaction *=p<0.01;**=p<0.05;

FIG. 21A-B. Effect of decreasing doses of MAIF on the ability ofneutrophils to emigrate from the vasculature into subcutaneouslyimplanted sterile sponges. p<0.01;

FIG. 22A-B. Effect of MAIF, administered at a dose of 20 mg per rat, toinhibit the ability of inflammatory cells to accumulate insubcutaneously implanted sponges when administered at the time ofimplant or up to 120 minutes after implant. *=p<0.01;

FIG. 23. Time course of the cellular inflammatory infiltration intosubcutaneously implanted sponges in normal animals;

FIG. 24. Effect of preparations of anti-inflammatory factor onplatelet-activating factor (PAF) induced adhesion of neutrophils tovenules;

FIG. 25. Effect of preparations of anti-inflammatory factor on PAFinduced neutrophil emigration;

FIG. 26. Effect of preparations of anti-inflammatory factor onPAF-induced flux of neutrophils through venules;

FIG. 27A-B. Reversal of neutrophil adhesion by preparations ofanti-inflammatory factor. FIG. 27A shows the effect of the MAIFpreparation (40 mg/rat) in reducing the number of neutrophils adheringto venules in response to PAF. FIG. 27B shows the effect of the MAIFpreparation (40 mg/rat) on new neutrophil endothelial cell adhesions;

FIG. 28. Effect of preparations of anti-inflammatory factor on thevelocity of neutrophils in venules;

FIG. 29. Effect of preparations of anti-inflammatory factor on thevelocity of red blood cells in venules;

FIG. 30. Effect of anti-inflammatory factor on leukocyte flux invenules;

FIG. 31A-B. Effect of 40 mg of the MAIF preparation administered i.v. onthe number of circulating neutrophils and lymphocytes in the 24 hoursfollowing injection;

FIG. 32. Dose-response relationship between i.v. administration of theMAIF preparation and circulating leukocyte numbers (p<0.01);

FIG. 33A-E. Effect of anti-inflammatory factor on various aspects oflymphocyte function. FIG. 33A shows the effect of prior administrationof factor on the response of host T lymphocytes to foreignhistocompatibility antigens. FIG. 33B shows the results obtained whenlymphocytes from MAIF treated rats are injected into untreated rats.FIGS. 33C-D show the effect of MAIF treatment on spleen weight andspleen cell number in rats. FIG. 33E shows the effect of MAIF treatmenton the concanavalin A stimulated mitogenic response of lymphocytes.

FIG. 34. Suppression of infection-induced edema by 40 mg of MAIFinjected i.v. The mean values of the two groups were: controls, 87±22μL; MAIF, 45±17 μL; p<0.01;

FIG. 35. Effect of MAIF given i.v. at 40 mg per rat on bacterialreplication and subcutaneously implanted, E. coli-infected sponges;

FIGS. 36A-B. Inhibition of inflammatory cell infiltration into infectedsponges by MAIF (40 mg per rat, i.v.);

FIG. 37. Effect of MAIF (40 mg per rat, i.v.) on suppression of theintermediate phase 4-16 hours) of inflammatory fluid accumulation in E.coli-infected sponges;

FIGS. 38A-C. Effect of 40 mg of MAIF, given intravenously at the time ofchallenge and 48 hours later, on the pathogenesis of experimentalpyelonephritis. The dotted line on the left-hand graph represents themean background kidney weight. *=p<0.01; **=p<0.02;

FIG. 39. Comparison of standardized hyperimmune and control MAIF MAIFprepared by standardized methods was tested at doses of 0.5, 1.5, 3, 5and 8 mg/120-150 gm. female rat for their ability to inhibit themigration of neutrophils to inflammatory sites. Reference to“commercial” is to off the shelf powdered skim milk;

FIG. 40. Comparison of MAIF and other milk components. The activity ofstandardized MAIF prepared from hyperimmune milk was compared withsialic acid and orotic acid, known components of milk believed to haveanti-inflammatory activity;

FIG. 41. Analysis of the composition of the DEAL-derived MAIF by sizeexclusion HPLC;

FIG. 42. Analysis of the dried ethyl acetate fraction in the neutrophilmigration inhibition assay compared to MAIF obtained prior to theorganic partition extraction. The dried ethyl acetate fractiondemonstrated strong MAIF activity;

FIG. 43. Analysis of hyperimmune whey and preparations of >1,000 daltonfiltrate from hyperimmune whey in the neutrophil migration inhibitionassay in comparison to >1,000 dalton filtrate prepared identically fromcontrol whey;

FIG. 44. Analysis of organic extracts of the >1,000 dalton filtrate onreversed-phase HPLC exhibiting a prominent peak at approximately 12minutes (Peak 12) as well as several other components;

FIG. 45. Analysis of fractions containing Peak 12 from reversed-phaseHPLC that have been pooled, lyophilized and rechromatographed onreversed-phase HPLC in a mobile phase containing only methanol andwater;

FIG. 46. Analysis of rechromatographed fractions containing Peak 12 inthe neutrophil migration inhibition assay. Peak 12 showed 60% to 80%inhibitory activity at doses of 3 to 30 nanograms;

FIG. 47. Analysis of rechromatographed fractions containing Peak 12 inthe neutrophil migration inhibition assay in comparison with aspirin,indomethacin and dexamethasone;

FIG. 48 illustrates one method of filtration in accordance with theprinciples of the present invention;

FIG. 49 illustrates one method for combining whey protein to the milkprior to ultrafiltration in accordance with the principles of thepresent invention;

FIG. 50 illustrates one method of filtration for combining whey proteinto the retentate subsequent to ultrafiltration in accordance with theprinciples of the present invention; and

FIG. 51 illustrates one method of ultrafiltration for obtaining >1000dalton retentate subsequent to ultrafiltration in accordance with theprinciples of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The invention comprises the isolation and purification of ananti-inflammatory factor from milk and the administration of said factorto an animal for the purpose of treating anti-inflammatory disorders.Except as otherwise indicted, the following definitions apply:

By the term “milk protein concentrate” (MPC) is any type of concentratedmilk product that contains 40-90% milk protein and/or any complete milk(casein plus lactalbumin) concentrate that is 40% or more protein byweight.

By the term “milk anti-inflammatory factor” is intended a factorobtained from either hyperimmune milk or normal cow's milk. By the term“substantially pure milk anti-inflammatory factor” is intended, for thepurpose of this invention, an anti-inflammatory factor that elutes as asingle major symmetrical peak on HPLC chromatography, after removal ofhigh molecular weight substances (>10,000 daltons) and isolation of thelow molecular weight, negatively-charged species by ion-exchangechromatography. By the term “substantially pure milk anti-inflammatoryfactor” is also intended anti-inflammatory factor in highly purifiedform produced by a process comprising: (i) ultrafiltering skimmed milkthrough a filter with a molecular weight cut-off of 1,000 daltons, (ii)collecting the >1,000 dalton retentate from step (iii) extracting theretentate from step (ii) by organic partition extraction and obtainingthe aqueous extract from the extraction; (iv) separating the aqueousextract from step (iii) by reversed phase HPLC chromatography; and (v)collecting the eluate. By the term “highly purified” or “highly pure” isintended an anti-inflammatory factor which has an elution patternsimilar, though not necessarily identical to that of FIG. 44, on areversed-phase HPLC chromatography column. Both normal milk andhyperimmune milk can be processed by the methods described herein toobtain the anti-inflammatory factor.

By the term “hyperimmune milk” is intended, for the purpose of thisinvention, milk obtained from milk-producing animals maintained in ahyperimmune state, the details for hyperimmunization being described ingreater detail below.

By the term “whey” is intended, for the purpose of this invention, milkfrom which cream has been removed.

By the term “normal milk” is intended for the purpose of the inventionmilk that is obtained from milk-producing animals by conventional meansand dairy practices.

By the term “milk-producing animal” is intended, for the purpose of thisinvention, mammals that produce milk in commercially feasiblequantities, preferably cows, sheep and goats, more preferably dairy cowsof the genus Bos (bovid), particularly those breeds giving the highestyields of milk, such as Holstein.

By the term “bacterial antigen” is intended, for the purpose of thisinvention, lyophilized preparation of heat-killed bacterial cells.

By the term “microencapsulated form” is intended, for the purpose ofthis invention, polymeric microparticles encapsulating one or morebacterial antigens for administration to milk-producing animals.

By the term “inflammation” is intended, for the purpose of thisinvention, a localized protective response elicited by injury ordestruction of tissues which serves to destroy, dilute or wall off boththe injurious agent and the injured tissue, characterized in the acuteform by the classical sequence of pain, heat, redness, swelling, andloss of function, and histologically involving a complex series ofevents, including dilatation of the arterioles, capillaries, and venuleswith increased permeability and blood flow, exudation of fluidsincluding plasma proteins, and leukocyte migration into the inflammatoryfocus.

By the term “treating” is intended, for the purposes of this invention,that the symptoms of the disorder and/or pathogenic origin of thedisorder be ameliorated or completely eliminated.

By the term “administer” is intended, for the purpose of this invention,any method of treating a subject with a substance, such as orally,intranasally, parenterally (intravenously, intramuscularly, orsubcutaneously), or rectally.

By the term “animal” is intended, for the purpose of this invention, anyliving creature that is subject to inflammation, including humans, farmanimals, domestic animals, or zoological garden animals.

Examples of inflammatory conditions that may be treated by the isolatedand purified milk product of the present invention are conditionsselected from the group consisting of acute and subacute bursitis, acutenon-specific tendinitis, systemic lupus erythematosus, systemicdermatomyositis, acute rheumatic carditis, pemphigus, bulbousdermatitis, herpeteformis, severe erythema, multiform exfoliativedermatitis, cirrhosis, seasonal perennial rhinitis, bronchial asthma,ectopic dermatitis, serum sickness, keratitis, opthalmicus iritis,diffuse ureitis, chorditis, optic neuritis, sympathetic ophthalmia,symptomatic sarcoidosis, Loeffler's syndrome, berylliosis, hemolyticanemia, mastitis, mastoiditis, contact dermatitis, allergicconjunctivitis, psoriatic arthritis, ankylosing spondylitis, acute goutyarthritis, and herpes zoster. Further, the isolated and purified milkproduct may be used to treat individuals who are exposed to potentiallyinflammatory agents.

The invention is based in part on the discovery that when amilk-producing animal such as a bovid is brought to a specific state ofhyperimmunization, the animal will produce milk which has supranormallevels of the highly beneficial anti-inflammatory factor, said factornot only suppressing the symptoms of inflammation in man and otheranimals, but al so being a prophylactic agent in anticipation of thepresence of inflammatory agents in the recipient. By the term“supranormal levels” is intended levels in excess of that found in milkfrom non-hyperimmunized animals. The induction of immune sensitivityalone is insufficient to cause the appearance of supranormal levels ofMAIF in milk, as is shown by the fact that normal cow's milk does notcontain these supranormal levels, even though the cows have becomesensitized against various antigens during normal immunization againstcow diseases and during normal exposure to the environment. It is onlyin specific hyperimmune states that the milk has the desired supranormallevels.

This special state may be achieved by administering an initialimmunization, followed by periodic boosters with sufficiently high dosesof specific antigens. The preferred dosage of booster should be equal toor greater than 50% of the dosage necessary to produce primaryimmunization of the bovid. Thus, there is a threshold booster dosagebelow which the properties are not produced in the milk, even though thecow is in what normally would be called an immune state. In order toachieve the requisite hyperimmune state it is essential to test thehyperimmune milk after a first series of booster administrations. If thebeneficial factors are not present in the milk, additional boosters ofhigh dosage are administered until the properties appear in the milk.

The process of producing the hyperimmune milk containing supranormallevels of anti-inflammatory factor is disclosed in co-pending U.S.patent application Ser. No. 580,382, filed Sep. 11, 1990 and also inU.S. Ser. No. 355,786, filed May 22, 1989 (now U.S. Pat. No. 5,106,618,a file wrapper continuation of U.S. Ser. No. 069,139, filed Jul. 2, 1987and in U.S. Ser. No. 910,297, filed Sep. 17, 1986 (now U.S. Pat. No.4,919,929, a file wrapper continuation of U.S. Ser. No. 576,001, filedFeb. 1, 1983); all of which are incorporated herein by reference intheir entirety. In summary, one process of producing the hyperimmunemilk containing supranormal levels of anti-inflammatory factor comprisesthe following steps: (1) antigen selection; (₂) primary immunization ofthe bovid; (3) testing the serum to confirm sensitivity induction; (4)hyperimmunization with boosters of appropriate dosage; and, optionally,(5) testing the milk for anti-inflammatory properties; (6) collectingthe milk from the hyperimmune bovid; and (7) processing the milk toisolate the MAIF.

Step 1: Any antigens or combination of antigens may be employed. Theantigens can be bacterial, viral, protozoan, fungal, cellular, or anyother substances to which the immune system of a milk-producing animalwill respond. The critical point in this step is that the antigen(s)must be capable, not only of inducing immune and hyperimmune states inthe milk-producing animal, but also of producing supranormal levels ofanti-inflammatory factor in the milk. Any antigen can be used to producesupranormal levels of factor. One preferred vaccine is a mixture ofpolyvalent bacterial antigens, referred to as Series 100 vaccine,described in detail in Example 1A below.

Step 2: The antigen(s) can be administered in any method that causessensitization. In one method, a vaccine composed of antigen derived from1×10⁶ to 1×10²⁰, preferably 10⁸ to 10¹⁰; most preferably 2×10⁸,heat-killed bacteria is administered by intramuscular injection.However, other methods such as intravenous injection, intraperitonealinjection, rectal suppository, or oral administration may be used.

Step 3: It is necessary to determine whether or not the milk-producinganimal has become sensitive to the antigen. There are a number ofmethods known to those skilled in the art of immunology to test forsensitivity (Methods in Immunology and Immunochemistry, William C. A.,and Chase, W. M., Academic Press, New York, vols. 1-5 (1975)). Thepreferred method is to use a polyvalent vaccine comprising multiplebacterial species as the antigen and to test for the presence ofagglutinating antibodies in the serum of the animal before and afterchallenge with the vaccine. The appearance of milk antibodies afterimmunization with the vaccine indicates sensitivity; at this point it ispossible to proceed to step 4.

Step 4: This involves the induction and maintenance of the hyperimmunestate in the sensitized animal. This is accomplished by repeated boosteradministration at fixed time intervals of the same polyvalent vaccinethat was used to achieve the primary sensitization. A two-week boosterinterval is optimal for polyvalent bacterial antigens. However, it isnecessary to ensure that the animal does not pass from a hyperimmunestate to a state of immune tolerance to the antigen.

In a preferred embodiment, hyperimmunization of bovids may be achievedby a single administration of microencapsulated vaccine, prepared asdescribed in detail in Example 1B below. The advantage of the controlledrelease form of hyperimmunization is that the constant exposure to theantigen ensures that the animal remains in the hyperimmune state.

In an alternative embodiment, it is also possible to combine differentimmunization procedures, e.g., simultaneously administeringmicroencapsulated and liquid antigen, or intramuscular injection forprimary immunization, and booster doses by oral administration orparenteral administration by microencapsulation means. Many differentcombinations of primary and hyperimmunization are known to those skilledin the art.

Step 5: It is necessary to test the milk for anti-inflammatory activitylevels. This can be accomplished by any research technique that teststhe effects of either the hyperimmune milk or products derived therefromupon inflammation. Chemical-induced inflammation of the rat paw is astandard assay for anti-inflammatory drugs.

Step 6: This involves the collection and processing of the milk. Themilk can be collected by conventional methods. Processing the milk toisolate the anti-inflammatory factor is described below.

The simplest process for isolating, purifying and testing theanti-inflammatory factor comprises the following steps:

1. defatting the hyperimmune milk to produce skim milk;2. ultrafiltration and diafiltration of skim milk to produce whey;3. optionally removing casein from skim milk to produce whey;4. removal from the whey macromolecules of molecular weight greater thanabout 10,000 daltons by ultrafiltration;5. fractionating the product from step 2 using an ion-exchange resincolumn to isolate a negatively-charged anti-inflammatory species;6. separating the negatively-charged species from step 4 by molecularsieve chromatography; and7. biological assay of the anti-inflammatory factor preparation fromstep 5.

In an alternative preferred embodiment, the fractions from molecularsieve chromatography that have biological activity are further purifiedby filtration through a membrane that retains macromolecules ofmolecular weight greater than about 5000 daltons.

Another preferred embodiment further comprises additional purificationof the by HPLC size exclusion chromatography and organic partitionextraction.

Yet another preferred embodiment of the present invention comprises thepurification of anti-inflammatory factor in highly purified formproduced by a process comprising: (i) ultratiltering skimmed milkthrough a filter with a molecular weight cut-off of 1,000 daltons;collecting the >1,000 dalton retentate from step (i); (iii) extractingthe retentate from step (ii) by organic partition extraction andobtaining the aqueous extract from the extraction; (iv) separating theaqueous extract from step (iii) by reversed phase HPLC chromatography;and (v) collecting the eluate

8. The anti-inflammatory action of the milk factor is tested on edemathat is caused by the injection of a solution of carrageenan into thepaw of rats. The rat paw test is the standard animal test foranti-inflammatory drugs. Winter, C. A Risley, G. A., Huss. A. W.,“Carrageenan-induced Edema in the Hind Paw of the Rat as an Assay forAnti-inflammatory Drugs,” Proc. Soc. Exper. Biol. Med. 3:544 (1967).Alternatively, one can use a pleural neutrophil migration inhibitionassay or tumor necrosis factor induction assay as described in Example24. Vinegar et al., “Some Quantitative Characteristics ofCarrageenan-induced Pleurisy in the Rat” Proc. Soc. Exp. Biol. Med. 143:711-714 (1973); Ammendola, G. et al. “Leukocyte Migration and LysozomalEnzymes Release in Rat Carrageenan Pleurisy,” Agents and Actions 5:250-255 (1975); Vinegar, R. et al. “Quantitative Studies of the Pathwayto Acute Carrageenan Inflammation.”. Fed. Proc. 35:2447-2456 (1976). Avariety of other tests may be used. Wetnick, A. S., and Sabin, C., “TheEffects of Clonixin and Bethaurethasone on Adjuvant-Induced Arthritisand Experimental Allergic Encephalomyelitis in Rats,” Jap. J Pharm.22:741 (1972). However, the rat paw test is the most simple and directtest available, and has been shown to be satisfactory for allanti-inflammatory drugs. This test has been described in detail in Beck,U.S. Pat. No. 4,284,623, which is incorporated herein by reference tothe extent that it describes the rat paw test. Briefly, the testinvolves the injection of a small quantity of carrageenan into thefootpad of adult white rats. This is known to induce an inflammatoryresponse. The resulting degree of swelling can be quantified. Samplescontaining an anti-inflammatory factor are administered to the rat by asuitable route, preferably by intraperitoneal injection, and theblockade or amelioration of the inflammatory process quantified byeither volumetric or gravimetric methods.

In summary, one can isolate the anti-inflammatory factor fromhyperimmunized milk by following a process of defatting the milk,ultrafiltration and diafiltration of skim milk to produce (MPC) MilkProtein Concentrate, and continuing with ion exchange and molecularsieve chromatography. The biological activity of appropriatepreparations of anti-inflammatory factor can be tested by doing adose-response experiment on rats as described herein.

In an additional preferred embodiment of the present invention, theanti-inflammatory factor present in hyperimmunized milk is purifiedusing a combination of steps involving: filtration on a membrane capableof separating molecules based upon their molecular weights; ion-exchangechromatography; molecular sieve chromatography; and affinitychromatography (Example 15).

The preferred first step comprises filtering hyperimmune skim milk,produced as described above, through a membrane which retains moleculeswith molecular weights of about 10,000 daltons or more. The materialpassed by the membrane (i.e. the filtrate or retentate) is collected andused in further purification steps. Devices and membranes for performingsuch filtrations are well-known in the art.

The preferred step following filtration is ion-exchange chromatographyon a anion exchanger. Exchangers having diethylaminoethyl groups havebeen found to effectuate good separations but it is expected that otheranion exchangers could be used as well. It is preferred that the solidsupport of the ion-exchanger be capable of maintaining high flow rates.Sepharose has been found to be suitable for this purpose.

The preferred step after ion-exchange chromatography is gel filtrationchromatography. A column packing for this step should be chosen which iscapable of fractionating molecules with molecular weights of less than10,000 daltons. The preferred packing is Toyopearl HW-40 (Rohm and Haas)but other packings well known in the art could be used as well. Examplesof other packings that could be used and which are commerciallyavailable are polymeric carbohydrate based packings, e.g. Sephadex G-10or G-25 (Pharmacia), or polyacrylamide based packings, e.g. Biogel P-2,P-4, P-6, P-10 or P-30, (Bio-Rad).

The preferred step after gel filtration chromatography is affinitychromatography on a boronate affinity support. These supports have beenfound to be effective at fractionating low molecular weight compoundswith cis-diol groups. The preferred support is AffiGel 601 (Bio-Rad).This is a boronate derivative of the polyacrylamide gel filtrationsupport Bio-Gel P-6 (also sold by Bio-Rad).

The preferred mode of storage for preparations after the ion exchange,gel filtration or affinity chromatography steps is as a lyophilizedpowder. The filtrate collected in the first purification step may bestored refrigerated until use. The activity of the anti-inflammatoryfactor resulting from the purification may be determined using the ratpaw test described above.

In another preferred embodiment of the present invention, theanti-inflammatory factor present in hyperimmunized milk is analyticallyor preparative purified using a combination of steps involving:ultrafiltrationion with a membrane having a molecular weight cutoff of10,000 daltons, ion exchange chromatography, size exclusionchromatography on a preparative column and organic partition extraction.

In a preferred first step, a large volume (e.g. 90 liter of skimmed milkis passed through an ultrafiltration membrane with a 10,000 daltoncutoff. The resulting retentate (<10K retentate) is collected forfurther purification steps.

The preferred step following the above ultrafiltration is applying alarge volume (e.g. 60 liters) of retentate from the previous step to anion exchange column. The eluate can then be lyophilized.Diethyl-aminoethyl (DEAE)-Sepharose Fast Flow exchange resin has beenfound to effectuate good separation.

The preferred step after the above ion-exchange chromatography isplacing on a preparative HPLC column a large quantity (e.g. 100 mg.) ofthe purified preparation from the ion-exchange column. Even largerquantities can be obtained by separating multiple samples of 100 mg eachon the HPLC column and collecting them into the same set of tubes. Thetubes can then be pooled and their contents lyophilized.

The next preferred step is organic partition extraction which entailstaking 50-100 mg. of the lyophilized HPLC column samples and extractingwith n-hexane to remove neutral lipids, acidification and thenreextraction with ethyl acetate.

In another preferred embodiment of the present invention,anti-inflammatory factor is purified by a process comprising: (i)ultrafiltering skimmed milk through a filter with a molecular weightcut-off of 1,000 daltons; (ii) collecting the =1,000 dalton retentatefrom step (i); (iii) extracting the retentate from step (ii) by organicpartition extraction and obtaining the aqueous extract from theextraction; (iv) separating the aqueous extract from step (iii) byreversed phase HPLC chromatography; and (v) collecting the eluate.

In a preferred step (i), the skimmed milk is in the form of hyperimmuneskimmed milk whey.

In a preferred step (ii), the organic partition extraction furthercomprises: (i) extracting with hexane and NH₄ OH; (ii) reextracting theaqueous phase from step (i) with ethyl acetate; and (iii) collectingsaid ethyl acetate extract.

In a preferred step (iii), the reversed-phase HPLC chromatography columnis a Zorbax SB-C₁₈ column.

Results of experiments described in Example 16 indicate thatpretreatment of animals with preparations of anti-inflammatory factorreduces the platelet activating factor (PAF) stimulated adhesion ofneutrophils to the endothelial cells which line venules and reduces therate at which neutrophils emigrate from venules. In addition, theadministration of preparations of the factor after treatment of animalswith PAF was found to reduce the number of neutrophils adhering toendothelial cells. To the extent that patients or animals may benefitfrom these effects, the present invention encompasses the use ofpreparations of the anti-inflammatory factor. This is true regardless ofthe particular disease involved. Similarly, the data in Example 16indicates that the anti-inflammatory factor causes its effects onadhesion and emigration by interacting directly with cell-surface CD18antigens and preventing other molecules from interacting with thisglycoprotein complex. The present invention encompasses the use ofpreparations of the anti-inflammatory factor for this purpose as well.

A shown in Example 18, the administration of a preparation ofanti-inflammatory factor to animals suppresses the Host vs. Graft butnot the Graft vs. Host reaction and causes an increase in spleen weightand in the number of splenic lymphocytes. The lymphocyte response toConcanavalin A was also found to be abrogated by the preparation. Thesedata indicate that the anti-inflammatory factor is useful in theinhibition of tissue destructive infectious processes, and in situationswhere suppression of lymphocyte function is desirable.

As shown in FIG. 42, desalting of the MAIF (obtained by organicpartition extraction) results in dramatic increases in purification asindicated by as much as a 10,000 fold lower dose of MAIF being requiredto obtain similiar levels of migration inhibition of rat pleuralleukocytes.

Accordingly, the present invention is also directed to methods for usinga milk anti-inflammatory factor, including a milk anti-inflammatoryfactor produced by any of the above alternative embodiments, to preventneutrophils from adhering to the endothelium of venules or to detachneutrophils which have already adhered to the endothelial cells liningthe walls of venules. In this way, the factor is used to reduce thetissue damage associated with the inflammatory response.

The invention is also directed to a method for using a milkanti-inflammatory factor, including the milk anti-inflammatory factor ofany of the above alternative embodiments to prevent interactions betweenCD18 cell-surface antigens and other molecules. It is known that suchinteractions are necessary for the exit of cells from the vasculatureand that such emigration leads to increased tissue damage in animalsduring the inflammatory response. CD18 antigens are also known to beimportant in the immunological response of a host organism to foreignantigens.

Also encompassed by the invention is the use of a anti-inflammatoryfactor, including the anti-inflammatory factor of any of the abovealternative embodiments, in mammals to prevent the emigration of cellsfrom the vasculature and to suppress the mitogenic response oflymphocytes to foreign antigens.

The compositions of the present invention may be administered by anymeans that provide anti-inflammatory activity. For example,administration may he parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal or oral.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms the activecompound is admixed with at least one inert diluent such as sucrose,lactose or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluent. In the case ofcapsules, tablets, and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared with anenteric coating.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsion, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the pharmaceutical art.Besides inert diluents, such compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or nonaqueous solutions, suspensions oremulsions. Examples of nonaqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils such as olive oil andinjectable organic esters such as ethyl oleate.

The dosage of active ingredients in the composition of this inventionmay be varied; however it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage form depends upon the desired therapeutic effect, on theroute of the administration and on the duration of the treatment.

Administration dosage and frequency will depend on the age and generalhealth condition of the patient, taking into consideration thepossibility of side effects. Administration will also be dependent onconcurrent treatment with other drugs and patients tolerance of theadministered drug.

Having now described the invention in general terns, the same will befurther described by reference to certain specific examples that areprovided herein for purposes of explanation only, and are not intendedto be limiting unless otherwise specified.

EXAMPLE 1A Preparation of S-100 Vaccine

A bacterial culture containing the spectrum of bacteria shown in Table 1below as obtained from the American Type Culture Collection wasreconstituted with 15 ml of media and incubated overnight at 37° C. Oncegood growth was obtained, approximately one-half of the bacterialsuspension was employed to inoculate one liter of broth with theinoculate being incubated at 37° C. The remaining suspension wastransferred to sterile glycol tubes and stored at −20° C. for up to sixmonths.

After good growth was visible in the culture, the bacterial cells wereharvested by centrifugation of the suspension for 20 minutes to removethe media. The bacterial pellet obtained was resuspended in sterilesaline solution and the bacterial sample was centrifuged three times towash the media from the cells. After the third sterile saline wash, thebacterial pellet obtained upon centrifugation was resuspended in a smallamount of double distilled water.

The media-free bacterial suspension was heat-killed by placing thesuspension in a glass flask in an 80″ C. water bath overnight. Theviability of the broth culture was tested with a small amount ofheat-killed bacteria. Broth was inoculated with heat-killed bacteria,incubated at 37° C. for five days and checked daily for growth, as thebacteria have to be killed for use in the vaccine.

The heat-killed bacteria were lyophilized until dry. The dry bacteriawere then mixed with sterile saline solution to a concentration of2.2×10⁸ bacterial cells/ml saline (1.0 optical density reading at 660nm).

TABLE 1 S-100 Bacteria List Gram Name Media + or − ATTC # 1. Staph.aureus BHI + 11631 2. Staph. epidermidis BHI + 155 3. Strep. pyogenes,A. Type 1 APT + 8671 4. Strep. pyogenes, A. Type 3 APT + 10389 5. Strep.pyogenes, A. Type 5 APT + 12347 6. Strep. pyogenes, A. Type 8 APT +12349 7. Strep. pyogenes, A. Type 12 APT + 11434 8. Strep. pyogenes, A.Type 14 APT + 12972 9. Strep. pyogenes, A. Type 18 APT + 12357 10.Strep. pyogenes, A. Type 22 APT + 10403 11. Aerobacter aerogenes BHI −884 12. Escherichia coli BHI − 26 13. Salmonella enteritidis BHI − 1307614. Pseudomonas aeruginosa BHI − 7700 15. Klebsiella pneumoniae BHI −9590 16. Salmonella typhimurium BHI − 13311 17. Haemophilus influenzaeBHI − 9333 18. Strep. mitis APT + 6249 19. Proteus vulgaris BHI − 1331520. Shigella dysenteriae BHI − 11835 21. Diplococcus pneumoniae APT +6303 22. Propionibacter acnes Broth + 11827 23. Strep. sanguis APT +10556 24. Strep. salivarius APT + 13419 25. Strep. mutans BHI + 2517526. Strep. agalactiae APT + 13813

Cows were given daily injections of 5 ml samples of the polyvalentliquid vaccine. Antibody (IgG) titer levels for the injected cattle weredetermined periodically by using an enzyme-linked immunoassay for bovineantibody against the polyvalent antigen.

EXAMPLE 1B Immunization Procedures

Heat-killed bacteria were prepared in the manner described above. Thepolyvalent antigen sample (S-100) obtained was microencapsulated by aconventional phase-separation process to prepare a polyvalentantigen-containing microparticle product. Generally, theantigen-containing shaped matrix materials are formed from polymers ofbiocompatible material, preferably biodegradable or bioerodablematerials, preferably polylactic acid, polyglycolic acid, copolymers oflactic and glycolic acids, polycaptolactone, copolyoxalates, proteinssuch as collagen, fatty acid esters of glycerol, and cellulose esters.These polymers are well known in the art and are described, for example,in U.S. Pat. No. 3,773,919; U.S. Pat. No. 3,887,699; U.S. Pat. No.4,118,470; U.S. Pat. No. 4,076,798; all incorporated by referenceherein. The polymeric matrix material employed was a biodegradablelactide-glycolide copolymer.

Heat-killed bacterial antigens are encapsulated in such matrixmaterials, preferably as microspheres of between 1-500 microns diameter,preferably 10×250 microns. The encapsulation processes are conventionaland comprise phase separation methods, interfacial reactions, andphysical methods. Many combinations of matrices and many concentrationsof assorted antigens may be employed, in order to provide for optimalrates of release of bacterial antigens to the host body from themicroparticles. These combinations can be determined by those skilled inthe art without undue experimentation.

The microparticles in the example were less than 250 microns indiameter. Approximately 750 mg of microparticles containing 22% (16.5mg) of polyvalent antigen was then suspended in about 3 cc of a vehicle(1 wt % Tween 20 and 2 wt % carboxymethyl cellulose in water).

A small group of cattle was selected from a larger herd of cattle. Fiveof these randomly selected cattle were selected as controls. Four cattlewere injected intramuscularly with microparticles containing polyvalentantigen. Microparticle samples were sterilized with 2.0 mRad of gammaradiation. Antibody (IgG) titer levels were determined periodically fromsamples of cows' milk obtained from the inoculated cows, as well as fromthe control cows.

EXAMPLE 2 Isolation of MAIF Factor from Hyperimmunized Milk Step 1: MilkFiltrate Preparation

Twenty liters of fresh milk from hyperimmunized cows were run through acream separator (DeLaval Model 102) to remove the fat.

The resulting sixteen liters of skimmed milk was ultra-filtered toremove the high molecular weight species (over 10,000 daltons) using ahollow fiber diafiltration/concentrator (Amicon DL-10L). Theconcentrator is equipped with two 10,000 daltons molecular weightcut-off cartridges (Ann con H₅ P₁₀₋₁₃). The skimmed milk was run at thepump speed of 80 on the meter and inlet and outlet pressure of 30 psiand 25 respectively.

Twelve liters of the filtrate (<10,000 daltons) coming out of thecartridges at the flow rate of four liters per hour was frozen orlyophilized for storage and for further purification.

Step 2: Ion-Exchange Chromatography

The milk anti-inflammatory factor, in the filtrate was first isolated byan anion exchange chromatography column.

In this procedure, DEAE-Sepharose CL-6B gel (Pharmacia) was used to packa 5×10 cm glass column which was equilibrated with sterile doubledistilled water, pH 7.0.

One liter of filtrate (<10,000) was applied to the column and elutedwith sterile double distilled water, pH 7.0 at the flow rate of 160 mlper hour. Ten milliliter fractions were collected and monitored at 280nm in an LKB Uvicord 4700 absorptiometer with an optical density printedout on a connected recorder (Pharmacia REC-482).

Substances other than the anti-inflammatory factor having positive andneutral charges are not bound to the DEAE-Sepharose gel. They are elutedat the fallthrough peak (first peak). The anti-inflammatory factorcarrying a negative charge is retained by the gel.

To elute the factor, the column was eluted with a stepwise gradientusing sterile physiological saline, pH 7.0. A typical profile is shownin FIG. 1. Bioassay of the individual fractions revealed that the secondpeak contains the factor. Fractions comprising the second peak and itsshoulder are used for further purification. Recovery studies show that8.8 grams of dried powder were obtained by this process.

Step 3: Gel Filtration Chromatography

The second peak obtained from Step 2 contains the anti-inflammatoryfactor and other negatively charged molecules; therefore, an additionalrefining step was needed. To achieve further purification, it isconvenient to use a gel filtration column to separate various componentson the basis of molecular weight.

In this process, Sephadex G-10 resin (Pharmacia) was packed into a2.5×80 cm glass column and equilibrated with sterile double distilledwater, pH 7.0. Two grams of the second fraction from Step 2 wasredissolved in sterile double distilled water and applied to the top ofthe column. The column was elated at the flow rate of 30 ml per hour.Fractions (3.3 ml) were collected and monitored at 254 nm and 280 nm(Pharmacia Duo Optical Unit) with optical density printed out on aconnected recorder (Pharmacia REC-482).

Typically, there were 3 peaks shown in the elution profile asillustrated in FIG. 2. The first and second peaks containedanti-inflammatory activity,

The first peak is an aggregate that forms on the G-10 column whichcontains the active factor.

The second peak contains the nonaggregated form of the factor. Both theaggregate form (peak 1) and the nonaggregated form (peak 2) arebiologically active in rat bioassay.

EXAMPLE 3 Characterization of Milk Anti-inflammatory Factor

The molecular weight of the non-aggregated form of factor prepared bythe method described above was found to be less than 10,000 daltons.This was deduced from the fact that the first step in the isolation ofthe factor from whey was by ultrafiltration using a membrane that doesnot allow the passage of molecular weight species >10,000 daltons.

The factor has a negative charge. This was determined by applying milkultrafiltrate to a DEAE cellulose ion exchange column. Theanti-inflammatory activity did not elute from the column with water.Changing the elution media to sodium chloride (0.9% pH) caused theelution of several peaks (FIG. 1). Neutral and positive charged speciesdo not adhere to the ion exchange resin, and negative charged speciesare eluted by increasing the salt concentration. When the less than10,000 dalton molecular weight retentate was applied to the DEAE column,neutral salts and sugars eluted with water (Peak 1, FIG. 1). Threedistinct peaks eluted when the buffer was changed to saline (Peaks 2-4).The second peak and its shoulder contained anti-inflammatory biologicalactivity in the rat assay. It is concluded, therefore, that the factorhas a negative charge.

Another chemical characteristic of the factor is that it forms anaggregate during the process of removing salt. This property becameapparent when <10,000 dalton molecular weight retentate was passed overa Sephadex G-10 column, equilibrated with double distilled water andeluted with water at a pH of 7 (FIG. 2). Three peaks eluted from theG-10 column; the first peak eluted with the void volume suggesting amolecular weight equal to or greater than 10,000 dalton. This wasunexpected because molecules greater than 10,000 daltons had previouslybeen removed from this sample by ultrafiltration. The second peak elutedin the position expected for the anti-inflammatory factor. Both thefirst and second peaks exhibited anti-inflammatory biological activityin the rat paw assay, whereas the third peak lacked activity. It wassurprising to find that both the first and second peaks hadanti-inflammatory biological activity. The material recovered from thefirst peak of the G-10 column (Step 3) was lyophilized and applied to aG-100 column; a single peak was eluted with the void volume, suggestinga molecular weight of 100,000 daltons or greater. The Step 3 G-10 columnremoves salt at the same time it separates the different molecularweight species. It is concluded, therefore, that during passage over theG-10 column and resulting removal of salt the anti-inflammatory factorformed a large molecular weight aggregate. The degree of aggregationvaried with the salt concentration.

The aggregation property suggests the possibility that a wide spectrumof different molecular weight species can be formed which haveanti-inflammatory biological activity due to the presence of theanti-inflammatory factor. The discovery of this property suggests thepossibility of producing milk anti-inflammatory factors having a widespectrum of different biochemical properties depending on the degree ofaggregation of the final product. For example, formulations havinglonger or shorter biological half lives might be produced by usinglarger or smaller molecular weight aggregates, with molecular weightdistribution being controlled by the salt concentration duringprocessing. The column chromatography method described herein results inthe smallest molecular weight species that has been obtained which hasbiological activity (i.e., peak 2 from the Step 3 G-10 column). Thisobservation also suggests using other methods for forming theaggregates. For example, dilution in water causes the aggregation tooccur. Chemical agents that bind salts, especially calcium can cause theformation of the aggregate. Having made this discovery, other methodsfor forming the aggregate and separating the factor will be obvious tothose skilled in the art.

EXAMPLE 4 Biological Activity Assay

The anti-inflammatory action of purified anti-inflammatory factor wastested on edema that was caused by the injection of a solution ofcarrageenan into the footpads of rats. A lyophilized sample of the milkanti-inflammatory factor preparation was dissolved in the appropriatevehicle and given intraperitoneally to experimental rats. Thecarrageenan was then administered to the rats in an amount of 0.1 ml ofa 1% saline solution in each hind footpad. The footpads were measuredbefore injections were given and 2.5 hours after the injections, using athickness gauge. The results are illustrated in Tables 2 and 3. In theseTables, the abbreviation MAIF refers to the preparation of milkanti-inflammatory factor obtained using the procedures described inExamples 1 and 2 above.

The non-aggregated form of the factor (peak 2 from the G-10 column) fromcontrol and hyperimmune milk caused reduction in inflammation of the ratpaw at doses between 1 mg and 25 mg (Table 2). Both the hyperimmune milkand the regular milk exhibited activity; however, the hyperimmunematerial was more potent. We concluded from this that theanti-inflammatory factor is present in greater concentration in the milkfrom hyperimmune cows.

The second peak from the DEAF column exhibited activity when isolatedfrom either hyperimmune milk or regular milk. The activity issubstantially greater in the hyperimmune milk (Table 3).

The first peak from the G-10 column, which is the aggregated form of thefactor, exhibited activity in rat paw tests (Table 2). However, theaggregated form is not as potent as the nonaggregated form on equalweight basis.

It is concluded from these studies that the anti-inflammatory factoroccurs naturally in cows milk. Hyperimmunization of the cows causeshigher concentration of factor in the milk. The factor is a small,negatively charged molecule that can be separated from the milk by avariety of methods. The factor can form large molecular weightaggregates that do not naturally occur in milk, but form duringprocessing.

TABLE 2 Effect of Milk Anti-Inflammatory Factor (MAIF) On Reduction ofInflammation in Rats Foot Pad Measurements (mm) Before After % MAIFDosage Injection Injection Difference Inflammation Prepared fromHyperimmune Milk 2.0 mg/rat 3.43 5.01 1.58 46 1.0 mg/rat 3.49 5.39 1.9054 0.5 mg/rat 3.42 5.51 2.09 61 0.1 mg/rat 3.43 5.86 2.43 71Control/saline 3.43 5.82 2.39 70 Prepared from Normal Cows Milk 2.0mg/rat 3.30 5.24 1.94 59 1.0 mg/rat 3.31 5.22 1.91 58 0.5 mg/rat 3.325.33 2.01 61 0.25 mg/rat 3.31 5.42 2.11 64

TABLE 3 Comparison of Semipurified Fractions of MAIF on Reduction ofInflammation in Rats (Prepared from Hyperimmune and Regular Milk) FootPad Measurements (mm) 2.5 hr. Before After % Injection InjectionDifference Inflammation DEAE Column 3.25 5.04 1.79 55 Second PeakHyperimmune Milk 2 mg/rat DEAE Column 3.30 5.24 1.94 59 Second PeakRegular Milk 2 mg/rat G-10 Column 3.31 4.98 1.67 50 First Peak 2 mg/ratControl/Saline 3.34 5.63 2.29 69

EXAMPLE 5 Chemical Analysis of Anti-inflammatory Factor

Anti-inflammatory factor samples were analyzed chemically. The factor isnot crystalline in structure, as determined by X-ray diffractionstudies. MAIF preparations gave an elemental analysis consistent withcarbohydrate composition. The C, H, O ratios were consistent with apolymeric or oligomeric material with some carbinol groups beingoxidized to carboxyl. The slight excess of calcium equivalents overchloride ions may be accounted for in part as carboxylate salts. Theremainder may be sodium or potassium salts. However, the meltingbehavior, or rather the non-melting behavior, was suggestive ofsalt-like and/or higher molecular weight compositions. The material inthe present state of purity apparently contains a variable amount ofsalts of calcium and chloride, probably CaCl₂.

Neither preparation contained a significant amount of nitrogen whichprecludes any peptide component in its composition. Likewise, theabsence of significant nitrogen can rule out the presence of aminosugars and other nitrogen-containing materials such as various complexlipids as the major component(s).

Pyrolytic mass spectra revealed significant traces of 18-carbon fattyacids. This fact, taken together with traces of N and P, suggest thepresence of a complex lipid in the preparation.

Infrared spectroscopy revealed absorptions consistent with carbinol andcarboxylate functionalities. Ultraviolet visible and fluorescentspectroscopy revealed no significant amount of chromophores beyond thoseindicated by infrared.

The chemical tests are consistent with an oligomeric carbohydrate,wherein the carbonyl function (aldehyde or ketone) is tied up in thesubunit linkages. The oligomeric carbohydrate also contains someside-chain oxidation to carboxylate.

The MAIF preparation is substantially, but not completely pure.

EXAMPLE 6 Rat Paw Edema Tests: Oral Administration

The rat carrageenan footpad assay was used to test the effectiveness ofthe anti-inflammatory factor as an in vivo anti-inflammatory agent.Thirty adult white rats were randomly divided into three groups of tenrats per group. The groups received, in five consecutive dailytreatments, either 10 mg of skim milk powder from hyperimmunizedanimals, 10 mg of skim milk powder from non-immunized animals or notreatment (20 ml water per day only). The powders were orallyadministered in 20 ml of water. On the fifth day the right paw of eachrat was injected with 0.1 ml of 1% carrageenan in saline. This procedureis known to cause acute inflammation (edema). Twenty-four hours afterinjection, the rats were sacrificed, the paws amputated, and the weightsof the left (control) and right (edematous) paws were compared. Theresults of the assay are shown in Table 4 (expressed as weight in grams)and in FIG. 3 (expressed as a percentage of the average weight ofcontrol paws).

TABLE 4 Rat Paw Edema Test Results (Paw wt, g, mean ± sem, n = 10)Carrageenan Control Difference Treatment Paw (wt, g) Paw (wt, g) (g)Immune Milk 1.78 ± 0.03 1.71 ± 0.02 0.06 ± 0.02 Control Milk 1.88 ± 0.061.64 ± 0.03 0.24 ± 0.05 Water 1.86 ± 0.03 1.65 ± 0.03 0.22 ± 0.02

The inflammatory response to carrageenan injection was markedly reducedin the immune milk treated rats as compared with the nonimmune milk andwater control groups. No evidence of side effects or adverse effects onthe general health of the rats was detected. From these data it can beconcluded that daily consumption of skim milk powder from hyperimmunizedanimals almost completely blocked the inflammatory response induced bycarrageenan injection in the footpad of rats.

EXAMPLE 7 Quantitative Rat Paw Edema Tests

A series of experiments was conducted on the hyperimmune milk fraction.The experiments were designed to confirm the anti-inflammatory activityof the milk anti-inflammatory factor when given intraperitoneally and toestablish a dose response curve, explore alternative routes ofadministration, and investigate dosage regimens which might form thebasis of further investigations.

Peak I from the G-10 column, supplied by Stolle Milk BiologicsInternational, was prepared according to the methods described in U.S.Pat. No. 4,956,349. Lactose, obtained from commercial sources, was usedas placebo. Aspirin was used as a positive control. Aspirin wasdissolved in water and given orally by gastric gavage at the ratio of200 mg per kilogram, a dose known to be active in the assay. A 2%solution of kappa carrageenan (Sigma C-1263) has been found to producethe most reproducible results and was thus used in these experiments.The footpad assay was modified by using isotopically labeled human serumalbumin (¹²⁵I-HSA) that localizes in the carrageenan-induced lesion indirect proportion to the volume of the exudate. By determining the totalradioactive count in the footpad and comparing this to the counts in aknown volume of plasma from the injected animal, a direct measurement ofedema in microliters of plasma equivalents is obtained. ¹²⁵I-HSA wasinjected intravenously at a dose of 1.0 microcurie per rat. Female DarkAgouti rats were used. The rats were approximately 12 weeks old, weighedbetween 160 grams and 200 grams, and were obtained from the in-houseinbred colony.

To conduct the carrageenan footpad assay, 0.1 ml of 2% carrageenan wasinjected subcutaneously into each hind foot pad of an anesthetized rat.This injection was followed immediately by injection of 1.0 microcurieof ²⁵¹I-HSA in 0.5 ml of saline into the tail vein. After four hours,each rat was weighed, blood samples obtained, and the rat euthanized.Both hind feet were then removed and the levels of radioactivity in eachfoot and in the 200 μl plasma standard were measured in an automatedgamma counter. From these measurements the volume of edema in each footwas calculated and expressed in microliters.

Experiment 1: Intraperitoneal Dose Response.

FIG. 4 illustrates the effect of intraperitoneal administration of apurified preparation of MAIF compared to lactose (CON), aspirin, and notreatment (No R_(x)). All treatments (lactose, aspirin, MAIF) were given30 minutes prior to the injection of carrageenan.

Carrageenan injection resulted in edema averaging 250 μl (No R_(x)). Theedema was inhibited by aspirin and all dosages of the MAIF preparationbut was not inhibited by lactose. The intraperitoneal dose-responsecurve obtained with the MAIF preparation, derived by expressing the dataas percentage of average control (no treatment) edema is shown in FIG.5.

Experiment 2: Effects of Various Routes of MAIF Administration.

FIG. 6 illustrates the effect, on footpad edema, of the administrationof lactose and a preparation of purified MAIF orally (ORAL),intramuscularly (IM), subcutaneously (SUB Q), and intravenously (IV).Also shown are a positive control (aspirin) and a nontreated control (NOR_(x)).

The preparations were administered prior to carrageenan challengeaccording to the following schedule: Aspirin: orally, 30 minutes prior;Subcutaneous MAIF: 1 hour prior; Oral MAIF: 24, 16 and 1 hour prior;intramuscular MAIF: 30 minutes prior; intravenous MAIF: at the time ofchallenge (isotope was also injected).

The results indicate that, expressed as the percentage of averagecontrol edema in each separate assay, the anti-inflammatory factor, byall routes of administration, inhibited edema formation. Fortymilligrams of the MAIF preparation given intravenously almost completelyabrogated the inflammatory response to carrageenan. These resultsdemonstrate the anti-inflammatory activity of MAIF and, in view of theresults of Experiment 1 above, suggest that the order of effectivenessfor different routes of administration is IV>IP>IM>SUB Q>ORAL.

Experiment 3: Effect on Edema of intravenous and Extended OralAdministration: Dose Response.

FIG. 7 shows the effects of IV and oral administration of a purifiedpreparation of anti-inflammatory factor on footpad edema in rats. MAIForal treatment (40 mg per rat per day) was given daily for six days andalso one hour before carrageenan challenge (PO). Intravenous treatments(5, 10, 20 mg) were given at the time of carrageenan challenge (IV).Also shown are a positive control (aspirin) and a negative control (notreatment).

The results shown in FIG. 7 indicate that all three dosages of the MAIFpreparation result in anti-inflammatory activity that exceeds even theactivity of aspirin in the assay, whereas extended oral administrationresults in marked but limited activity.

The study was therefore extended to examine the effects of furtherreduced intravenous dosages of anti-inflammatory factor. Intravenousdosages of lactose placebo were included as a control. The results ofthese studies are shown in FIG. 8. Intravenous dosages of 2.5 and 1 mgof the MAIF preparation (IV) induced anti-inflammatory activity in therange of the activity induced by aspirin. 10 ml of intravenous lactoseplacebo (10 mg PLAC IV) did not induce activity in that range.

An intravenous dose-response curve was derived by combining the resultsof Experiments 2 and 3 and expressing these results as percentageaverage control edema (no treatment) in each separate assay. The curveis shown in FIG. 9,

The conclusions that may be drawn from the quantitative rat paw edematests are as follows: milk fraction peak I from the G-10 column,extracted and purified as described in U.S. Pat. No. 4,956,349,consistently shows anti-inflammatory activity when tested in the rat pawedema model. A dosage of 4 mgs of MAIF preparation per rat givenintravenously at the time of carrageenan injection is sufficient todrastically inhibit edema and was therefore chosen as a standard againstwhich other preparations would be compared in further experiments.

EXAMPLE 8 Anti-Inflammatory Properties of Preparations of HyperimmuneMilk Obtained from Identical Twin Cows

The effect of vaccination on the anti-inflammatory activity of milk wasinvestigated by testing the bioactivity of various milk fractionsobtained from identical twin cows. Based on the extraction methodsdescribed in U.S. Pat. No. 4,956,349 an extraction scheme utilizingultra-filtration was devised. The processing sequence was as follows:##STR1##.

Milk samples were prepared from immunized twin cows, non-immunizedcontrol twin cows, and reconstituted skim milk powder previouslyprepared from immunized cows. The sample group consisted of 45 sets ofidentical twin cows. One cow of each twin set was vaccinated bi-weeklywith Stolle S100 mixed bacterin (described in U.S. Pat. No. 4,956,349).The bioactivity of the various fractions was tested by intravenousinjection using the rat carrageenan footpad assay described above.

The hypotheses to be tested were that (a) hyperimmunization wasresponsible for the anti-inflammatory activity described above. (b) MAIFcould be extracted on a commercial scale by ultra-filtration and (c)dilution of the retentate would cause aggregation of theanti-inflammatory factor, causing it to be retained by the 30,000molecular weight ultra-filtration membrane.

FIG. 10 illustrates the results of a twin herd ultra-filtrationexperiment designed to test the bioactivity of various fractions madefrom the milk of non-vaccinated control twins and from reconstitutedmilk powder from immunized cows. The fractions that were tested are asfollows: Peak I, G-10 column preparation, 4 mls (OHIO MAIF STD); R₂final retentate from non-vaccinated twin (CONTROL TWIN R₂); P₂ finalretentate from the reconstituted milk powder (RECON S100 P₂); dialyzedR₂ final retentate from non-vaccinated twin (CON DIALYZED R₂); dialyzedfinal retentate from the reconstituted milk powder (S100 DIALYZED R₂),

No anti-inflammatory activity could be detected in the R₂ finalretentate fraction prepared from nonimmunized cows, even after dialysis.No anti-inflammatory activity was detected in the final retentateP₂fraction prepared from the reconstituted milk powder. Thereconstituted milk powder retentate R₂ fraction, following dialysis,exhibited anti-inflammatory activity in the range of the activity of theMAIF standard.

FIG. 11 illustrates the results of twin herd ultra-filtrationexperiments designed to test the bioactivity of various mi fractionsmade from vaccinated and non vaccinated twin cows and from reconstitutedmilk powder from immunized cows. The fractions that were tested are asfollows: Peak I, G-10 column preparation, 4 ml (OHIO MAIF STD); dialyzedfinal retentate R₂ from non-vaccinated twins (CON DIALYZED R₂); finalretentate R₂ from the reconstituted milk powder (RECON S100 R₂); thefinal retentate R₂ from vaccinated twins (IMMUNE TWIN R₂); firstretentate R1 from the reconstituted milk powder, diluted for: 1 (S100DILUTED R1).

Little anti-inflammatory activity was detected in the dialyzed retentateR₂ from non-vaccinated control twins or in the non-dialyzed retentateR₂from the vaccinated twins. Some activity is detectable by scattergram.R₂ retentate prepared without dialysis from reconstituted Stolle milkpowder from immunized cows was strongly anti-inflammatory. However, thepreparation in made by dilution of the reconstituted milk beforeultrafiltration rather than dilution of whey made from the milk was onlymarginally active. This result indicates that anti-inflammatory activityis more efficiently extracted from the whey fraction.

FIG. 12 illustrates the results of twin herd ultrafiltration experimentsdesigned to test the bioactivity of dialyzed retentate from vaccinatedtwin cows. The fractions tested are as follows: Peak I, G-10 columnpreparation (OHIO MAIF STD); dialyzed final retentate R₂ from vaccinatedtwins (IMM DIALYZED R₂); dialyzed final retentate from the G-10preparation (DIALYZED OHIO MAIF). The results show thatanti-inflammatory activity was present in the R₂ fraction from theimmunized twin after dialysis. Dialyzed MAIF was more active in theassay than the nondialyzed MAIF standard. This result suggests thatdialysis is an effective means of further concentrating the milk factorresponsible for anti-inflammatory activity.

The results presented in FIGS. 10-12 above support the followingconclusions: (1) anti-inflammatory activity can be extracted fromreconstituted milk from immunized cows by ultrafiltration of the dilutedretentate. (2) anti-inflammatory activity was not demonstrated in theabove-preparations that were made from the milk of non-immunized cows.(3) anti-inflammatory activity was demonstrated in the final retentateR₂ after ultrafiltration of diluted retentate prepared from the milk ofimmunized cows, but dialysis was necessary in order to demonstrate theactivity.

EXAMPLE 9 Stability of MAIF, Heating, and Proteinase Treatment of MAIF

The previous evidence that the milk anti-inflammatory factor waschemically not a protein or a peptide was based largely on chemicalanalyses that consistently showed an almost complete absence ofnitrogen. For further characterization of the anti-inflammatory factor,several preparations were tested in the rat paw edema assay, using 4 mgsof peak I, G-10 column preparation, intravenously as the standard. Thefollowing treatments were done: proteinase (pronase) treatment for sixhours; six hours no proteinase treatment control; untreated positivecontrol; heating at 100° C. for 30 minutes.

The results of this assay are illustrated in FIG. 13. The conclusionsderived from this study were that the anti-inflammatory activity is notdue to a protein or peptide and that the anti-inflammatory factor is notinactivated by boiling. The effectiveness of pronase treatment wasverified by the finding that parallel pronase treatment completelydenatured milk protein.

EXAMPLE 10 Anti-Inflammatory Activity of Further Purified MAIF and WheyProtein Concentrate from Immunized Cows

Retentate and retentate from ultrafiltration using an Amicon YM5membrane were tested for biological activity using intravenousadministration in the rat paw edema assay. In this process, the MAIF inpeak I of the G-10 column, prepared according to U.S. Pat. No.4,956,349, was further purified by ultrafiltration on an Amicon YM5membrane. This membrane retains molecules of 5000 molecular weight orgreater. Whey protein concentrates (WPCs) were also prepared from milkfrom immunized animals and filtered through the YM5 membrane. Thefollowing samples were tested in the assay using 4 mg peak I, G-10column preparation, intravenously as the standard: retentate from AmiconYM5 ultrafiltration; retentate from Amicon YM5 ultrafiltration; WPC fromimmunized cows, 30 mgs per rat; WPC from commercial production(non-immunized cows), 30 mg per rat.

The results of this assay are illustrated in FIG. 14. It is clear fromthese results that all of the activity is in the retentate whichcomprised approximately 0.5% of the total weight of the fraction appliedto the YM5 filter. The reduction of edema seen in this experiment wasachieved following administration of 20-25 micrograms of material.

Regarding the activity of WPC, WPC made from hyperimmunized animalsdearly showed anti-inflammatory activity as expected. Interestingly, WPCmade from non-immunized animals also showed anti-inflammatory activity.The presence of anti -inflammatory activity in the milk of nonimmunizedcows is not surprising since it must be a natural substance. Itsdetection reflects the sensitivity of the bioassay.

EXAMPLE 11 Continuous Monitoring of Carrageenan Induced Footpad Edema

It was established that 4 mg of MAIF preparation given intravenously atthe time of carrageenan injection reduced the accumulation of edema inthe footpad by between 40% and 50%. Although these results providedevidence that the material contained an anti-inflammatory moiety, therewas little indication of the site of action or pharmacological profileof MAIF. In order to obtain such data it was necessary to establish amethod that allowed the continuous monitoring of footpad edemathroughout the response to carrageenan. This was achieved by holding therat foot in a demounted Gamma radiation detector. The procedure requiredanimals to be anesthetized for up to four hours and, as anesthetics areknown to suppress the inflammatory response, it was first necessary todetermine the effect of anesthetics on the carrageenan-induced edema.Five agents commonly used to induce anesthesia in rats were thereforeevaluated; these were ether, chloral hydrate, Innovar-vet, nembutal andurethane. The results are shown in FIG. 15.

It was clear from these results that ether was the anesthetic of choicewhen the inflammatory response was to be evaluated by this technique.The shape of the curve obtained when ether was used indicated a biphasicresponse. To delineate the response in more detail a further experimentwas carried out in which the volume of edema was measured at 12 timepoints over a 5 hour period. The results confirmed a biphasic response.The early response occurred between 0 and 1 hour after challenge andlate phase response between 1.5 and 2 hours (FIG. 16).

The two phases, which have also been observed by other investigators,have been termed the non-phagocytic inflammatory response (NPIR) and thephagocytic inflammatory response (PIR), respectively.

The NPIR is initiated, in response to injury, by soluble mediators suchas histamine and bradykinin while the PIR depends on the participationof neutrophils. The protocol, therefore, was to administer MAIF andmonitor the accumulation of edema continuously in an effort to determinewhether the anti-inflammatory properties of the agent were a result ofan effect on the early non-cellular (NM) or the later cellular (PIR)phase. 5 mg or 40 mg of MAIF preparation per rat were administeredintravenously at the time of carrageenan challenge and the accumulationof edema monitored at regular intervals over a four hour period. Neitherdose affected the accumulation of edema during either phase (FIGS.17A-B).

This result was surprising as many previous analyses, in which theeffect of purified preparations of MAIF on carrageenan induced edema 4hours after challenge was determined, had demonstrated considerableanti-inflammatory activity in the fractions. It was likely, therefore,that the continuous exposure to ether suppressed or inactivated theactive anti-inflammatory component of MAIF in vivo.

Previous studies indicated that short term exposure to ether did notaffect the activity of the anti-inflammatory factor. Therefore, anexperiment was done in which the effect of MAIF on progressive edemaaccumulation was determined at only four time points, O, 1, 3 and 4hours, thus limiting the exposure of the animals to ether. The 1 hourtime point was chosen to assess the affect on the early non-phagocyticinflammatory response while the 3 and 4 hour measurements were selectedto quantify the effect on the later phagocytic inflammatory response. Inthis experiment the MAIF preparation administered at 40 mg resulted in areduction in the accumulation of edema during the secondary,phagocytic-cell mediated phase, but had no significant effect on theprimary, soluble mediator driven phase (FIG. 18).

The following conclusions can be drawn from this series of experiments.

1. Ether is the preferred anesthetic for use in experiments where theinflammatory response to carrageenan is to be monitored continuously.2. Continuous ether anesthesia inhibits the in vivo anti-inflammatoryactivity of anti-inflammatory factor in the carrageenan footpad assay3. MAIF ameliorates inflammation by inhibiting the late, phagocytic-cellmediated phase of the inflammatory response to carrageenan.

EXAMPLE 12 Time Course of the Effect of MAIF on Carrageenan InducedFootpad Edema

A further series of experiments were carried out in which the agent wasadministered at selected time points before or after the injection ofcarrageenan rather than at the time of challenge. The purpose of thestudy was to provide information on

(a) the most effective time for administration of MAE in relation to theinflammatory stimulus.(b) the biological half life of the anti-inflammatory moiety.(c) the points in the development inflammatory response affected byMAIF.

The study was carried out in three parts. A preparation of MAIF wasadministered intravenously at a dose of 4 mg/rat at one of 11 timepoints, ranging from 150 minutes before, to 150 minutes after injectionof carrageenan. Results of this experiment are shown in FIG. 19 andTable 5.

TABLE 5 Inhibition Time of A Mean foot Mean foot of edema in relationvolume of volume of by MAIF Experi- to challenge control groups MAIFgroups (% of control ment (min) (μl ± SD) (μl ± SD) volume ± SD) 3 −150311 ± 65 246 ± 52 79 ± 17 2 −90 304 ± 71 211 ± 33 73 ± 11 2 −60 304 ± 71186 ± 34 61 ± 11 1 −30 391 ± 63 261 ± 49 67 ± 13 3 −15 311 ± 65 152 ± 4149 ± 13 1,2,3 0 336 ± 78 184 ± 42 55 ± 13 3 15 311 ± 65 218 ± 30 70 ± 101 30 391 ± 63 218 ± 30 56 ± 8 2 60 304 ± 71 212 ± 40 69 ± 13 2 90 304 ±71 216 ± 37 70 ± 12 3 150 311 ± 65 261 ± 42 84 ± 14

A significant inhibition of edema was observed at all time pointsstudied; however, the level of inhibition was less at the outer extremes(±150 min). An interesting cyclic response to MAIF administration wasseen in those groups treated closer to the point of challenge. The factthat MAIF was more effective when given 30 minutes after challenge thanwhen given 15 minutes after challenge supports the concept that thesecondary, phagocytic-cell mediated, phase of the response is inhibitedby the agent. The preparation of MAIF strongly inhibited the response tocarrageenan when administered 15 minutes before or at the time ofchallenge. It is apparent, furthermore, that the agent has a relativelylong half life in the serum (1-2 h) and its effectiveness is related tothe time of challenge and the dynamic nature of the inflammatoryresponse.

It is thus surmised that the anti-inflammatory effect is due to aneffect on inflammatory cells, likely the neutrophils.

EXAMPLE 13 Effect of MAIF on the Reverse Passive Arthus Reaction

The possibility that the anti-inflammatory factor might affectneutrophil involvement was investigated by evaluating the ability of thematerial to modulate the reverse passive. Arthus reaction (RPA). Thisimmune complex-induced response is primarily neutrophil mediated andagents which affect the development of the reaction do so via an effecton these cells. To induce the RPA, rats were injected intradermally withrabbit antibody to ovalbumin and intravenously with native ovalbumin.Ovalbumin/ovalbumin-antibody immune complexes form in and around thedermal blood vessel walls, host neutrophils bind to the Fc portion ofthe antibody and an intense inflammatory reaction is initiated. Itshould be noted that, although the response is initiated byimmune-complexes, it takes place independently of the host's immunesystem.

Three parameters are used to quantify the RPA. These are, (1)edema—measured using the accumulation of ¹²⁵I-HSA, (2)hemorrhage—assessed by in vivo pre-labelling RBC's with ⁵⁹Fe and (3)neutrophil accumulation—measured by determining tissue levels of theneutrophils specific enzyme myeloperoxidase (MPO). These assays areknown to those of ordinary, skill in the art.

Eighteen rats were divided into three groups of six. Rabbitanti-ovalbumin (40 μl) was injected intradermally at four sites on thehack of each animal and 2 mg of ovalbumin injected intravenouslyimmediately afterwards. One group of animals received no other treatmentand served as controls. The second group were injected intravenouslywith 20 mg of a lactose preparation, while the final group were injectedintravenously with 20 mg of a purified preparation of MAIF. Both lactoseand MAIF preparation were administered with the ovalbumin. The severityof the reaction was assessed 3.5 hours after challenge. When the MAIFpreparation was administered intravenously at a dose of 20 mg/rat priorto the initiation of the RPA response, there was a highly significantinhibition of the three parameters used to measure the response (TABLE6, FIG. 20A-C). The lactose control material also caused a modest andmarginally significant suppression of neutrophil accumulation andhemorrhage. This indicates that there is a small amount ofanti-inflammatory activity in normal milk.

TABLE 6 Neutrophil accumulation: Haemorrhage: Group Units of MPO μl ofEdema μl of RBC Control 0.30 ± .157 107 ± 29 4.8 ± 3.1 Lactose 0.214 ±.176** 104 ± 23 3.0 ± 1.5** MAIF 0.056 ± .013 60 ± 27* 1.5 ± 1.7* * = p< 0.01 ** = p < 0.05

As the neutrophil is the primary mediator of the RPA, these resultsprovided additional evidence that MAIF was capable of inhibiting theinflammatory response via an effect on neutrophil function.

EXAMPLE 14 Effect of MAIF on Neutrophil Migration

In order to participate effectively in an inflammatory response,neutrophils must first migrate from the vasculature to the site ofinflammation. To determine whether anti-inflammatory factor interferedwith neutrophil migration, a model of inflammation employing thesubcutaneous implantation of sterile polyurethane sponges was used. Thesponges are removed at intervals after implantation and by weighing thesponges and then extracting and counting the cells in the infiltrate,both the fluid and cellular phase of the response can be quantified.Twenty four hours after implantation >95% of the cel is found in thesponge are neutrophils.

Two experiments have been carried out. In the first, animals weretreated with either 5, 10, 20, or 40 mg of a purified MAIF preparationat the time of sponge implantation. Sponges were removed 24 hours afterimplantation. Each group consisted of between 5 and 8 rats and twosponges were implanted in each animal. The results are shown in FIG.21A-B.

Twenty or 40 mg of MAIF preparation, administered intravenously at thetime of sponge implantation, had a marked effect on the ability ofinflammatory cells to migrate. A less marked, but equally significant,inhibition of fluid accumulation was also seen. The two lower doses ofMAIF had no demonstrable effect in this model of inflammation.

A second experiment, designed to delineate the temporal relationshipbetween the inflammatory challenge (sponge implantation) and MAIFadministration, was carried out. In this study, 20 mg of MAIFpreparation were administered intravenously 30, 60 or 120 minutes aftersponge implantation. A fourth, control, group was left untreated. Therewere five animals in each group. Two sponges were implanted in eachanimal and these were removed after 24 hours. The results areillustrated in FIGS. 22A-B. Included on this graph are results obtainedfrom a sample group of rats that received 20 mg of the MAIF preparationat the time of implantation (see FIG. 21A-B).

Results from the time-course of the effect of MAIF oncarrageenan-induced footpad edema show MAIF to be comparativelyineffective when administered 60 minutes or later after challenge. It isnoteworthy that while 20 mg of the MAIF preparation was required tosuppress the inflammation associated with the sponge implantation, 4 mgwas sufficient to inhibit the carrageenan-induced edema. Withoutintending to be held to this interpretation, this disparity may berelated to the different level of provocation presented to the host bythe two stimuli. The sponge implant is a relatively benign stimuluswhich induces a slow inflammatory response and the bulk of the cellsaccumulate between 8 and 16 hours after implantation (FIG. 23). On theother hand the subcutaneous injection of carrageenan is a very strongstimulant which induces a correspondingly strong response over arelatively short period (FIG. 16).

EXAMPLE 15 Alternative Method of a Purifying the Anti-InflammatoryFactor from Milk (Preparation “AIF ”)

The following Example describes a method for purifying theanti-inflammatory factor from milk in its lowest molecular weight,unaggregated form. The preparation resulting from the purification stepsdescribed herein has been given the designation “AIF” in order todistinguish it from the preparation obtained using the proceduredescribed in Example 2. In the present Example and in the Example whichfollows (i.e. Example 16) the active factor within the preparations isreferred to simply as the “anti-inflammatory factor”. All of thepurification steps were performed so as to minimize possiblecontamination with bacteria or pyrogens. Sterile water was used toprepare solutions and all glassware was depyrogenated.

Step 1: <10,000 Molecular Weight (“MW”) Ultrafiltration

Fresh S100 immune skim milk (see Example 1 for a description ofprocedures used in obtaining the immune milk) was pumped through a10,000 MW cutoff ultrafiltration membrane (Filtron) at a pressure of 30psi. The retentate was collected in depyrogenated bottles maintained onice. Retentates were sterile filtered and refrigerated until use. The<10,000 MW retentate contains the milk anti-inflammatory factor as wellas low molecular weight peptides, oligosaccharides and a large amount oflactose. Anti-inflammatory activity in the retentate occurs in a lowmolecular weight, unaggregated form.

Step 2: DEAE-Sepharose Chromatography

Initial fractionation of anti-inflammatory activity was performed onDEAE-Sepharose. A 5×50 cm column containing one liter of DEAE-Sepharosewas equilibrated with retentate buffer. Retentate buffer is a sterile,endotoxin-free solution containing the diffusible ions found in bulkmilk in the appropriate concentrations. Retentate buffer contains CaCl₂,MgCl₂, NaCl, NaCitrate and NaH₂ PO₄. Typically, approximately eightliters of <10,000 MW retentate were pumped onto the DEAE-Sepharosecolumn at a flow rate of about 500 ml per hour. Column eluate wasmonitored at 280 nm. The column was washed with distilled water untilthe 280 absorbance returned to baseline (about 6 to 8 liters ofdistilled water were typically required). Anti-inflammatory activity wasbound to the column and was eluted with about 4 liters of 0.5 M.ammonium acetate in water, pH 7.4. The eluate was lyophilized to drynessand weighed. The weight of recovered material obtained from eight litersof retentate was typically between 15 and 20 grams. Since ammoniumacetate is completely volitized during lyophilization, the residualweight represents the weight of bound material. Anti-inflammatoryactivity was assayed in the mouse neutrophil migration inhibition assay.

Step 3: H-40 Chromatography

The material eluted from the DEAE-Sepharose column was furtherfractionated on a sizing column in order to separate the factorresponsible for anti-inflammatory activity from other low molecularweight components. Eight grams of the DEAE sample was dissolved in 50 mlof distilled water and applied to a 2.5×150 cm column containing 736 mlof Toyopearl HW-40 (Rohm and Haas) equilibrated in water. The column wasdeveloped in distilled water at a flow rate of 40 ml per hour and eluatewas monitored at 280 nm. Fractions were collected and assayed foranti-inflammatory activity in the mouse neutrophil migration inhibitionassay. Fractions evidencing, activity and minimal absorbance at 280 nmwere pooled and lyophilized. Approximately 80 mg of material containinganti-inflammatory activity was recovered from eight liters of retentate.

Step 4: AffiGel 601 Chromatography

The final purification step involved affinity chromatography of theactive factor in a column packed with a boronate-derivatizedpolyacrylamide based medium (AffiGel 601, Bio-Rad) which has an affinityfor coplanar adjacent cis hydroxyl groups. Forty mg of low molecularweight HW-40 derived material was equilibrated in 10 ml of 0.25 Mammonium acetate, pH 7.0, and applied to the AffiGel column which hadalso been equilibrated in 0.25 ammonium acetate. Eluate was monitored at280 nm. The column was washed with 400 ml of 0.25 M ammonium acetate ata flow rate of 50 ml per hour until the 280 nm absorbance decreased tobackground. The AffiGel column was then eluted with 1600 ml of 0.1 Mformic acid, pH 2.8. The eluate was tested for activity in the mouseneutrophil migration inhibition assay and lyophilized to dryness.Approximately 8 to 10 mg of bound material containing anti-inflammatoryactivity was recovered from 8 liters of retentate.

The preparation obtained by this method is given the designation “ADP”.The preparation was substantially purified with respect to theanti-inflammatory factor but is not homogeneous. The preparationexhibits anti-inflammatory activity in the mouse neutrophil migrationinhibition assay, in the rat paw edema assay, in the rat ear swellingassay and blocks neutrophil binding to rat mesentery venule endothelium(visualized by intravital microscopy). Based upon comparative analysesin the mouse neutrophil migration inhibition assay, AIF is approximately55,000 fold more purified than the original skim milk <10,000 MWretentate.

EXAMPLE 16 Effect of Preparations of Anti-inflammatory Factor on theAdhesion of Neutrophils to Endothelial Cells and on the Emigration ofNeutrophils from the Vasculature

The effect of the anti-inflammatory factor on the adhesion ofneutrophils to endothelial cells and on the emigration of neutrophilsfrom the vasculature was tested. Two different preparations ofanti-inflammatory factor were used. One preparation was made using thepurification procedure described in Example 2. For the purposes of thepresent Example, this preparation is referred to simply as “MAIF”. Theother preparation of anti-inflammatory factor was made using thepurification procedure described in Example 15 and is referred to bothin that Example and in the present Example as “AIF”. It is to beunderstood that both MAIF and AIF contain within them theanti-inflammatory factor at different states of purity.

Chemicals:

Human serum albumin, trypsin, platelet-activating factor (PAF), phorbolmyristate acetate (PMA), propidium iodide, and Histopaque were Obtainedfrom Sigma Chemical Co., St. Louis, Mo. Human Neutrophil elastase waspurchased from Calbiochem. A murine anti-human CD18 monoclonal antibody(IgG₁-subclass; FITC conjugate) and a murine anti-keyhole limpethemocyanin (IgG₁-subclass; FITC conjugate), used as a negative controlantibody, were purchased from Becton Dickinson Systems Inc., MountainView, Calif. Simply Cellular™ Microbeads were purchased from FlowCytometry Standards Corp., Research Triangle Park, N.C. Other reagentswere the best grade commercially available and were used without furtherpurification.

In Vivo Methods:

Intravital microscopy experimentation. Twenty-four male Wistar rats(180-250 g) were maintained on a purified laboratory diet and fasted for24 hr prior to surgery. The animals were initially anesthetized withpentobarbital (12 mg/100 g body weight). A right carotid artery andjugular vein were cannulated to measure systemic arterial pressure(Statham P23A pressure transducer and a Grass physiologic recorder) anddrug administration respectively. A midline abdominal incision was madeand the animals were placed in a supine position. A segment of therind-jejunum was exteriorized through the abdominal incision and allexposed tissue was covered with saline soaked gauze to minimize tissuedehydration. The mesentery was carefully placed over an optically clearviewing pedestal that allowed for transillumination of a 2 cm² segmentof tissue. The temperature of the pedestal was maintained at 37° C. witha constant temperature circulator (Fisher Scientific, model 80). Rectaland mesenteric temperatures were monitored using an electrothermometer.The mesentery was suffused with warmed bicarbonate-buffered saline (pH7.4). An intravital microscope (Nikon Optiphot-2 Japan) with an X25objective lens (Leitz Wetzlar L25/0.35, Germany) and X10 eyepiece wasused to observe the mesenteric microcirculation. A video camera mountedon the microscope projected the image onto a color monitor and theimages were recorded for playback analysis using a video cassetterecorder. Single unbranched venules with diameters ranging between 25and 40 μm were selected for study. Venular diameter was measured on lineusing a video caliper. The n umber of adherent and emigrated neutrophilswas determined off-line during playback of videotaped images. Aneutrophil was considered adherent to venular endothelium if it remainedstationary for 30 seconds or more. Rolling neutrophils were defined asthose white blood cells that moved at a velocity less than that oferythrocytes in the same vessel. Leukocyte rolling velocity wasdetermined by the time required for a leukocyte to traverse a givendistance along the length of the venule.

Experimental protocol. After all hemodynamic parameters were in steadystate, images from the mesentery were recorded for 5 minutes. Themesentery was then superfused for 60 minutes with 100 nM PAF in thepresence of either 40 or 5 mg/rat of the MAIF preparation (iv.).Measurements of aforementioned parameters were again performed at 30 and60 min of PAF superfusion. In two experimental groups, the mesentericpreparations were again exposed to PAF as described above but, at 30minutes, they received either 40 or 5 mg/rat of the MAIF preparation. Inthree additional experiments, the AIF preparation was given either as apretreatment or as a post-treatment.

In Vitro Methods

Isolation of Neutrophils. Neutrophils from healthy donors were purifiedby dextran sedimentation followed by hypotonic lysis and Histopaquecentrifugation. Except for the dextran sedimentation step, which wasperformed at room temperature, the cells were kept at 4° C. throughoutthe isolation procedure. Cell preparations contained 95% neutrophils andgreater than 99% of these were viable as determined using Trypan Blue.After isolation, neutrophils were resuspended at a final concentrationof 2×10⁶ cells/ml in phosphate buffered saline (PBS). Aliquots of cellswere then incubated at 37° C. for 20 minutes with varying concentrationsof either the MAIF or the AIF preparation. After washing, neutrophilswere incubated in the dark at 4° C. for 30 minutes with saturatingconcentrations of fluorescein-conjugated murine anti-human CD18, humanCD11b, IGG coated microbeads (Simply Cellular™ microbeads) or the murinenegative control antibody.

Immunofluorescence Staining and FACS Analysis. Direct immunofluorescenceas a measure of CD18 surface expression was determined by analysis on aFACScan (Becton Dickinson Systems Inc., Mountain View, Calif.) using thechannel number (log scale) representing the mean fluorescence intensityof 10,000 cells. The logarithmic channel numbers were converted tolinear values using methods well-known in the art. The specific meanfluorescence intensity for cells stained by CD1.8 antibodies wascalculated after subtracting the mean fluorescence intensity of thecells exposed to the negative control antibody. Non-viable cells werescreened out using propidium iodide.

Superoxide Assay. Superoxide production from isolated neutrophils wasmeasured following PMA and N-formyl-Met-Leu-Phe (“fMLP”) stimulation inthe presence of various concentrations of MAIF. The reduction ofcytochrome C by activated neutrophils was measured using aspectrophotometer (Hitachi U2000) at 550 nm. Briefly, sample was addedto two cuvettes and one cuvette was used as a reference. The lattercontained superoxide dismutase (superoxide scavenger). Neutrophils wereallowed to equilibrate at 37° C. for 5 min in the presence of variousconcentrations of MAIF and the cells were then stimulated with eitherPMA or FMLP. Superoxide production was measured for 3 min.

Protease Release. ¹²⁵I-labelled albumin was coated onto wells andallowed to dry overnight. Unbound albumin was washed and thenPMA-stimulated neutrophils were incubated within the wells for one hourin the presence or absence of various concentrations of MAIF. Freeradioactivity within the supernatant of the wells was divided by totalradioactivity within each well to assess the level of proteolysis.

Results:

Results are summarized in FIGS. 24-30 and Tables 7-9. FIG. 24demonstrates that PAF superfusion increased neutrophil adhesion topostcapillary venules approximately 6-fold over a 60 min period. 40mg/rat of the MAIF preparation reduced the PAF-induced neutrophiladhesion by more than 90% at 30 minutes and by more than 80% at 60minutes. Interestingly, MAIF pretreatment seemed to also reduce thenumber of adherent neutrophils prior to exposure of PAF. The lowerconcentration of MAIF (5 mg per rat) was less effective, reducingleukocyte adhesion by 50% at 60 min. The AIF preparation at aconcentration of 0.01 mg per rat was found to reduce leukocyte adhesionby about 50% at 60 min. At a tenfold higher concentration of ALF, a verylarge increase in leukocyte adhesion was observed (data not shown). Theadhesion was so dramatic that the videotape could not be analyzed. FIG.25 shows the effect of MAIF and AIF on neutrophil emigration. MAIF at aconcentration of 40 mg per rat and 5 mg per rat and AIF at aconcentration of 0.01 mg per rat were found to completely prevent theincrease in neutrophil emigration with time of PAF exposure. Neutrophilflux did not appear to change significantly in the MAIF treated groupcompared with the untreated group (FIG. 26). When AIF was given, weinitially observed more neutrophils rolling than usual, however thenumber decreased with time.

In a second series of experiments, the various anti-inflammatory agentswere administered after neutrophils were already adherent (FIG. 27A-B).In this series of experiments, leukocyte adhesion was reversed by anMAIF dose of 40 mg/rat but not by a dose of 5 mg/rat. AIF at a dose of0.01 mg per rat reversed neutrophil adhesion by approximately 25%. Tofurther assess the effect of the higher concentration of MAIF (40mg/rat), the number of adherent neutrophils at the start of therecording procedure and the number of new neutrophils that adhered over5 min at each period were examined. FIG. 27A demonstrates that therewere fewer neutrophils adherent following 10 min. of MAIF administrationindicating that the anti-inflammatory factor had actually “peeled off”adherent neutrophils. Moreover, FIG. 27B clearly demonstrates that MAIFblocked new neutrophil-endothelial cell adhesions. The speed with whichneutrophils rolled along the length of venules did not change betweengroups or with time with the exception that AIF may have increasedneutrophil rolling velocity (FIG. 28). This effect was ratherinteresting in light of the fact that red blood cell velocity remainedunchanged (FIG. 29). The results suggest that a simple increase inhydrodynamic forces cannot explain the increase in neutrophil rollingvelocity. Neutrophil flux also was unaffected by MAIF but was againreduced by AIF (FIG. 30).

In vitro data indicates that the anti-inflammatory factor does notinterfere with the activation of neutrophils per se. The superoxideradical scavenger, superoxide dismutase completely blocked cytochrome creduction by PMA and fMLP-stimulated neutrophils, suggesting that thisis a superoxide-mediated process. MAIF at extremely high concentrationsonly minimally affected cytochrome c reduction, suggesting that MAIFdoes not directly scavenge superoxide (Table 7). Protease release wasnot affected by MAIF (data not shown).

It was found that the binding of anti-CD18 monoclonal antibody could bereduced with MAIF or AIF (Table 8). This did not occur with the CD11bantibody. Binding of CD18 antibody to IgG coated microbeads was also notaffected by the MAIF or AIF preparations suggesting that theanti-inflammatory factor was not affecting the ability of the anti-CD18monoclonal antibody to bind to substrate but was, more likely, actingupon the ligand, CD18. The same pattern was observed with stimulatedneutrophils (Table 9). It should be noted that the binding to CD18varied between days because different cells were used each day.Therefore, a direct comparison of the results in Table 8 with those inTable 9 cannot be made.

TABLE 7 Effect of MAIF on Superoxide Secretion by Cells PMA-StimulatedfMLP-Stimulated Superoxide Production Superoxide Production MAIF(nmole/min/10⁷ cells) (nmole/min/10⁷ cells) 0.0 mg/ml 153 55 0.1 mg/ml145 50 1.0 mg/ml 143 40 5.0 mg/ml 140 32 10.0 mg/ml 127 —

TABLE 8 Effect of Anti-Inflammatory Factor on the Availability of CD18and CD11 Cell Surface Antigens Mean Channel Mean Channel UnstimulatedFluorescence Fluorescence Neutrophils Anti-CD18 Antibody Anti-CD11Antibody Neutrophils alone 314.24 1594.57 +0.1 mg/ml MAIF 234.26 1553.74+1.0 mg/ml MAIF 262.78 1796.00 +0.1 μg/ml AIF 248.28 1577.04 +1.0 μg/MLAIF 188.93 1554.61 Beads + Anti- CD18 Antibody 60.03 +1 mg/ml MAIF 88.61+1 mg/ml AIF 84.99

TABLE 9 Effect of MAIF on the Availability of CD18 Cell Surface Antigenson Stimulated and Unstimulated Neutrophils Mean Channel FluorescenceUnstimulated Neutrophils 236.95 + MAIF 1 mg/ml 216.08 + MAIF 5 mg/ml251.51 Stimulated Neutrophils 266.69 + MAIF 1 mg/ml 158.68 + MAIF 5mg/ml 171.96

Discussion:

The data in the above Example suggests that the anti-inflammatory factorprevents neutrophil adhesion and emigration in venules in adose-dependent manner. More importantly however, the anti-inflammatoryfactor could, within a brief period (10 min), reverse neutrophiladhesion to these vessels. The only other agents that cause adherentneutrophils to release their hold on the endothelium with suchefficiency are monoclonal antibodies directed against the CD11/CD18glycoprotein complex on the neutrophil. MAIF did not appear to have anyeffect on blood flow through the individual vessels or on systemic bloodpressure, suggesting that hemodynamic factors such as shear stress couldnot account for the reversal of leukocyte adhesion. Although leukocyterolling appears to be a prerequisite for leukocyte adhesion, MAIF didnot effect leukocyte rolling velocity or leukocyte flux. The latterresult suggests that the anti-inflammatory factor did not affect thenumber of neutrophils that rolled through the vessel and therefore, thatthe reduction in adherent leukocytes was not a result of fewerleukocytes interacting with the endothelium. The fact that leukocyterolling velocity as well as leukocyte flux remained unchanged suggeststhat adhesion molecules on neutrophils and endothelium responsible forleukocyte rolling (1L-selectin-selection) were not affected by theanti-inflammatory factor in the MAIF preparation.

It has been reported that the leukocyte may regulate its own adhesion byreleasing superoxide as well as proteases. It was therefore conceivablethat the lack of leukocyte adhesion in the presence of MAIF and AIF wasdue to the ability of these preparations to block the release superoxideor proteases. This possibility is untenable in light of the fact thatMAIF had little effect on superoxide or protease release and did notinteract with released proteases or scavenge released superoxide.Moreover, the MAIF did not appear to affect neutrophil viability asassessed with propidium iodide making a direct cytotoxic effect of theanti-inflammatory factor on neutrophils unlikely.

For a neutrophil to adhere and emigrate it must have an intact CD11/CD18glycoprotein complex. Immunoneutralization of the adhesion complexcompletely impairs the ability of the neutrophil to permanently adhereto the endothelium and emigrate into the surrounding tissue. Sinceneutrophil adhesion and emigration is a rate limiting step in the tissueinjury associated with a number of inflammatory conditions, an agentthat interferes with these processes would also likely block theinflammatory response. In the present study, both MAIF and AIFdramatically reversed neutrophil adhesion and blocked neutrophilemigration induced by PAF. Because of the similarity between AIF-, MAIF-and anti-CD18 monoclonal antibody induced reversal of neutrophiladhesion, it seemed possible that the anti-inflammatory factor withinAIF and MAIF exerted its effect by directly interacting with the CD18glycoprotein complex. The in vitro data presented above supports thisview, in that both AIF and MAIF blocked the ability of an anti-CD18antibody to bind to the CD18 glycoprotein complex. In contrast, neitherAIf nor MAIF affected the binding of CD11b to its respective monoclonalantibody. Finally, the AIF and MAIf preparations did not interfere withthe ability of the anti-CD18 monoclonal antibody to bind to IgG-coatedmicrobeads. Therefore, it can be concluded that the anti-inflammatoryfactor interacts with the CD18 complex directly and prevents CD18 frombinding to various ligands, including endothelial cell adhesionmolecules.

EXAMPLE 17 Effect of MAIF on Circulating Leucocytes

Several pharmacological agents can inhibit neutrophil migration. Whilesome, such as cyclophosphamide, are cytoreductive and act by inhibitinghemopoiesis in the hone marrow, other agents, such as steroids and thenon-steroidal anti-inflammatory drugs, have specific sites of action anddo not result in leucocytosis. It was important therefore to determinethe effect of the anti-inflammatory factor on circulating white bloodcell numbers and ratios.

Two experiments were done. In the first, the MAIF preparation wasadministered intravenously at a dose of 40 mg/rat to one group of 6animals and a control group was injected with saline. Blood samples wereobtained at baseline, 1, 4, and 24 hours after treatment. The resultsare summarized in FIG. 31A-B.

MAIF administration resulted in an increase in circulating neutrophilnumbers, maximal at 4 hours, and a corresponding decrease in the numberof peripheral blood lymphocytes. A further dose-response study wascarried out in which a group of rats were injected intravenously withsaline, 5, 10 or 20 mg of the MAIF preparation. Blood from each rat hadbeen taken 7 days previously to provide baseline values and was takenagain 4 hours after the injection of MAIF. The results are shown in FIG.32. Included on the graph are the results obtained from the sample taken4 hours after the administration of 40 mg the MAIF preparation (see FIG.31).

All doses of MAIF resulted in an increase in the number of circulatingneutrophils and a decrease in the number of lymphocytes. While theeffect on lymphocytes was linearly related to dose, the increase inneutrophil numbers was in the form of a curve, the greatest effect beingobserved in those animals given 10 mg.

These results support the concept that the anti-inflammatory factormodulates inflammation by affecting the adhesion of neutrophils toendothelial cells.

Data were also obtained pertaining to the effect of three othercell-targetted, anti-inflammatory/immunomodulatory agents on circulatingleucocytes in the rat. The steroidal drug, methylprednisolone, causes achange in the lymphocyte/neutrophil ratio analogous to that seen withMAIF. The temporal relationship between drug administration and effectis somewhat different. The anti-rejection/anti-inflammatory agentcyclosporin A also causes an increase in the number of circulatingneutrophils but lymphocyte numbers are either increased or not affecteddepending on the dose. In contrast, the cytotoxic drug cyclophosphamidedepletes both circulating lymphocytes and neutrophils. The effects ofthe anti-inflammatory factor would appear to closely parallel the actionof methyl-prednisolone.

EXAMPLE 18 Effect of the Anti-inflammatory Factor on Lymphocyte Function

The ability of the anti-inflammatory factor to induce a reversibledecrease in the number of circulating lymphocytes (Example 17) promptedfurther investigation of the effect of the factor on lymphocytefunction. Graft versus Host (GvH) and Host versus Graft (HvG) analyseswere used to determine the effect of the factor on T lymphocytefunction.

In the HvG analysis, parental Dark Agouti rats (“DA”) were injected i.v.with 20 mg of the MAIF preparation 48, 24 and 3 hours before lymphocytesfrom their F1 hybrid offspring (DA×Hooded Oxford rats) were injectedinto their footpads. Thus, the effect of the anti-inflammatory factor onthe ability of T lymphocytes from an intact host (DA) to respond to theforeign histocompatibility antigens of the F1 lymphocytes was measured.The protocol produced a highly significant reduction (30%) in theresponse as evidenced by a decrease in popliteal lymph node weight (FIG.33A).

In the GvH reaction parental (DA) lymphocytes were obtained from MAIFtreated parental rats (DA) and injected into the footpads of their F1(DA×Hooded Oxford) offspring. This assay measured the in vivoresponsiveness of I lymphocytes removed from the host under evaluation,i.e. from MAIF treated rats. The MAIF regimen had no effect on the GvHresponse (FIG. 33B).

During the preceding experiments, an apparent increase in the number ofsplenic lymphocytes in MAIF treated animals was noted. Furtherexperiments showed a significant increase in both spleen weight and inspleen cell numbers (FIGS. 33C and 33D). The increase in spleen cellnumbers was approximately equal to the decrease in the number ofcirculating cells reported previously.

Finally, the effect of the anti-inflammatory factor on the ability ofisolated splenic lymphocytes to respond to the mitogen concanavalin Awas determined. Administration of the MAIF preparation was found toalmost totally abrogate the mitogenic response of cultured lymphocytesto this lectin (FIG. 33E).

EXAMPLE 19 Suppression of Infection Induced Inflammation by theAnti-inflammatory Factor

Experiments have been carried out to determine whether changes in serumlevels of acute phase reactants (APRs) could be used to quantify theanti-inflammatory activity of the anti-inflammatory factor. The APRs area group of proteins which are synthesized in response to an inflammatorystimulus. One of these, alpha 2 macroglobulin, is common to both man andrats and methodology for measuring this inflammatory component isavailable. Two intravenous injections of MAIF preparation (0 and 24hours) did not reduce the peak response (48 hours) of alpha 2macroglobulin. This result indicates that the factor does not affect thelater inflammatory response.

EXAMPLE 20 In Vitro and in vivo Evaluation of Milk DerivedAnti-Inflammatory Factor (Bovine Mammary Macrophage Assay, InfectionModels in Mice)

Incubation of bovine mammary macrophages with the hyperimmune milkfraction did not detectably enhance the degree of phagocytosis but didincrease the ability of macrophages to kill phagocytosed Staphylococcusaureus. Mice injected intraperitoneally with 10 mg of the MAIFpreparation per kilogram demonstrated increased resistance tointraperitoneal challenge with lethal Staphylococcus aureus.

In an intra-mammary Staphylococcus aureus mastitis challenge model, MAIFinjected mice also showed significantly less mammary inflammation andinvolution and increased clearance of the infectious organism.Quantitative histological analysis of mammary tissue from MAIF treatedmice showed significantly more lumen, less interalveolar connectivetissue, and less leukorytic infiltration compared to control mice.Mammary glands of treated mice also contained fewer colony forming unitsthan control mice. The anti-inflammatory appears to exert its effect onthe non-specific defense system by a modulation of leukocyte function.

EXAMPLE 21 Effect of the Anti-Inflammatory Factor on the Pathogenesis ofExperimental Infection

The most common inflammagens encountered by man are microbial and it isimportant to determine the effect of any agent which modulates hostdefenses against infection. The tissue damage which accompanies manyinfectious diseases is in fact caused by the host response to infectionrather than by the invading organism. While the ability to modulate theinflammatory response to infection could be a useful clinical technique,it must be recognized that inhibition of the host response duringinfection can be disadvantageous. This is especially true in the case ofneutrophil inhibition. Studies with agents which curb the participationof neutrophils in the early stages of infection have demonstrated that,while inflammation and tissue damage may be initially suppressed, theincreased bacterial load that occurs as a result of the reduced cellularresponse eventual leads to an exacerbation of tissue damage. Thus, it isessential to evaluate the potential of the milk anti-inflammatory factorto modulate infection in order to (1) determine if the agent can reduceinfection-induced tissue damage and (2) to assess whether any observedsuppression of the host response is accompanied by an increase in theseverity of infection.

The effect of the anti-inflammatory factor on edema formation followingthe intradermal injection of E. coli 075 was determined. Two groups of 8animals were used. One group was untreated and served as controls whileindividuals in the second group were injected intravenously with 40 mgof the MAIF preparation in 0.5 ml saline. Immediately after theadministration of MAIF, 100 μl of an overnight culture of E. coli 075was injected intradermally at two skin sites on the shaved back of therat, followed by the intradermal injection of 100 μl of saline at twofurther sites. To allow estimation of edema volume in the infected skin,0.1 μCi of ¹²⁵I-HSA was injected intravenously at the time of challenge.Six hours later the animals were anaesthetized, a blood sample obtained,the skin on the back removed and the infected and saline injected sitespunched out. The volume of edema was calculated by relating tissuecounts to plasma counts as described. To obtain the volume of edemawhich accumulates as a result of the presence of E. coli theedema/plasma volume of the saline-injected sites was subtracted. Theresults are shown in FIG.

MAIF administration resulted in a 48% inhibition of edema formation.This experiment established that the anti-inflammatory factor couldmodulate the local inflammatory response to infection.

In order to study the relationships between anti-inflammatory factoradministration, bacterial replication, the accumulation of fluid andinflammatory cell infiltration, an alternative model of infection wasemployed. Polyurethane sponges, prepared and implanted as previouslydescribed, were infected with a quantitated sample of E. coli 075 at thetime of implantation. The sponges were removed at timed intervals,weighed to determine the volume of the fluid exudate, and then squeezedin media to free the bacteria and cells from the sponge. Bacterial andcell numbers were estimated using techniques known to those skilled inthe art. The following experiment was carried out using this model.Ninety animals were divided into two groups of 45. One of these groupswas untreated and served as controls. The second group were injectedintravenously with 40 mg of the MAIF preparation. The sponges were thenimplanted subcutaneously and, at the time of implantation, each spongewas inoculated with 10⁵ E. coli 075. Groups of 6-8 animals were killedat intervals thereafter and the bacteriological status and the size ofthe inflammatory infiltrate in the sponges determined. The results areillustrated in FIGS. 35-37.

The rate of bacterial replication was much greater in MAIF treatedanimals than in the controls and there was a 10, 1000 and 10,000 folddifference in bacterial numbers at 4, 8 and 16 hours respectively.Thereafter, bacterial numbers declined, although there was still a largedifference at 96 hours (FIG. 35).

The early response to infection is the critical determinant in theoutcome of an infectious episode. In this experiment the cellularinfiltrate at 2, 4 and 8 h in those animals given MAIF was 27%, 35% and46% of the control infiltrate respectively (FIG. 36A-B). The cells whichaccumulate in the first 24 h after challenge are >90% neutrophils andthe suppression of this cellular component during this phase may accountfor the rapid increase in bacterial numbers. The accumulation of fluidat 2 hours was not affected by the administration of MAIF, but wassignificantly less 4, 8 and 16 hours following challenge. This isconsistent with the previous finding that the anti-inflammatory factordid not suppress the primary, non-cellular phase of edema formation inthe carrageenan footpad model. In previous studies, using theimmunomodulatory agents cyclosporin A and methylprednisolone, a similarassociation between the suppression of the acute cellular inflammatoryinfiltrate and the promotion of bacterial replication was shown.However, in these experiments, the increased bacterial load promoted ahost response between 24 and 48 hours post challenge in which there wasa massive influx of neutrophils. When tissue was involved, the enhancedinflammatory response resulted in a marked exacerbation of tissue damageand scar formation. Interestingly, although administration of MAIFsuppressed the early inflammatory response and was associated with a10,000 fold increase in bacterial numbers there was no massive influx ofneutrophils 24-48 hours post challenge.

EXAMPLE 22 Effect of the Anti-Inflammatory Factor on ExperimentalPyelonephritis

An agent which can suppress inflammation in infection without resultingin a sequela of enhanced tissue damage would have considerablepotential. A clinically relevant model of infectious disease couldprovide an experimental basis for establishing such potential.

Pyelonephritis is an infectious disease which demonstrates localinflammation, tissue destruction and scar formation as cardinalhistological features. A well characterized model of the disease isavailable, which reproduces the central pathological features of thedisease in man. Pyelonephritis is induced in the rat by the directinoculation of the surgically exposed kidney with a predetermined numberof E. coli 075. Following challenge, bacterial numbers increase rapidlyand reach a peak 3 to 4 days later. In normal animals the level ofinfection declines over the following 5 or 6 days and reaches a plateauat about 10 days post challenge. By 21 days the lesions have resolvedand present as focal areas of indented scar tissue. To assess the effectof the anti-inflammatory factor on this model of infection,pyelonephritis was induced in both kidneys of twenty-six animals. Onehalf of these animals were treated with the MAT preparationintravenously at a dose of 40 mg/rat at the time of challenge and again48 hours later. Seven animals from each group were killed 4 days afterinduction of pyelonephritis and the two remaining groups of six animalsat 21 days. Kidneys were removed aseptically and weighed to determinethe relative volume of the fluid exudate. The extent of the surfacelesion size was estimated by direct visualization and the kidneyhomogenized to allow the enumeration of bacterial numbers. The resultsare shown in FIG. 38A-C.

Four days after challenge the inflammatory response, as evidenced by theinhibition of fluid accumulation and the size of the lesions on thesurface of the kidney, was suppressed by the administration of MAIF. Aspreviously observed in the studies involving infected,subcutaneously-implanted sponges, the early suppression of inflammationresulted in a logarithmic increase in the number of bacteria inMAIF-treated animals. By 21 days there was no difference in thepathology of disease as measured by kidney weight, bacterial numbers orrenal surface lesions size. Thus, while suppression of the earlyinflammatory response with MAIF did not result in a reduction in tissuedestruction in the chronic (21 day) phase of pyelonephritis, neither didit promote the development of pathological lesions as otheranti-inflammatory and immunomodulatory agents have done.

EXAMPLE 23 Summary of Experimental Data

A method was developed which allowed the accumulation of edema in thecarrageenan injected footpad to be monitored continuously.

The early, non-phagocytic, phase of the inflammatory response was notaffected by anti-inflammatory factor, whereas the later,cellular-driven, phase of the reaction was significantly inhibited.Further experiments, in which MAIF was administered at intervals beforeor after the injection of carrageenan, provided additional evidence thatMAIF exerted its anti-inflammatory effect by modulating the secondary,neutrophil-mediated, inflammatory response.

The anti-inflammatory factor was shown to have a half-life of 1-2 hoursfollowing i.v. injection and development of inflammation could besuppressed when the factor was administered 30 minutes after challenge.This result is relevant to the potential therapeutic use of theanti-inflammatory factor.

The neutrophil is the principal cell involved in the acute inflammatoryresponse. During the Arthus reaction, a >80% reduction in neutrophilaccumulation was observed following MAIF administration which, in turn,was associated with a highly significant inhibition of the secondarycharacteristics of the inflammatory reaction, namely edema andhemorrhage. This result further implicated neutrophils as a target inMAIF-induced suppression of inflammation.

One of the key steps in the development of inflammation is the migrationof neutrophils from the vasculature to the tissue. The intravenousadministration of the anti-inflammatory factor was shown to result inprofound and dose dependent inhibition of neutrophil migration. When theeffect of the anti-inflammatory factor on peripheral blood leukocyteswas investigated, a marked increase in the number of circulatingneutrophils was observed, accompanied by a corresponding decrease in thenumber of lymphocytes. This effect was also dose-dependent, but in thecase of the increase in neutrophil numbers, was not linear.

The administration of the milk and-inflammatory factor was found to bothblock the adhesion of neutrophils to the endothelium and to promote thedissociation of hose neutrophils which were adherent at the time ofadministration. This effect is probably the result of the ability of theanti-inflammatory factor to block the interaction between cell surfaceCD18 antigens and other molecules. The inhibition of CD18 binding by thefactor appears to be specific in that the factor prevented the bindingof anti-CD18 monoclonal antibody to cells but did not similarly preventthe binding of anti-CD11b monoclonal antibody.

The blocking of intermolecular interactions involving the CD18 cellsurface antigen may also account for the observation that the factor wasable to inhibit the ability of host lymphocytes to respond to foreignhistocompatibility antigens. In other experiments, the anti-inflammatoryfactor was found to block the concanavalin-induced mitogenic response inlymphocytes.

Finally, the factor significantly suppressed the early cellular responseto infection, an effect which resulted in a logarithmic increase inbacterial numbers in a model of subcutaneous infection. Thisexacerbation of infection did not result in a rebound of theinflammatory response, as seen with other agents which suppress acuteinflammation in infection. A second experiment using a clinicallyrelevant model of infection, pyelonephritis, also demonstrated asuppressive effect on inflammation which was associated with an increasein bacterial numbers. Again no rebound effect was observed and there wasno difference in the degree of tissue damage which occurred in the MAIFtreated and control groups.

The following conclusions can be drawn from this series of experiments:

1. Anti-inflammatory factor, administered i.v. suppresses the secondary,neutrophil-mediated, phase of the carrageenan induced inflammatoryresponse.2. When evaluated in the carrageenan footpad assay the anti-inflammatoryfactor has a biological half-life of 1-2 hours and is effective evenwhen administered after inflammation is induced. Subsequent experimentsindicate that the effective half-life is dependent on both the dose andinflammatory stimulus employed.3. Anti-inflammatory factor inhibits neutrophil emigration in vivo.4. Anti-inflammatory factor administration results in an increase in thenumber of circulating neutrophils and a corresponding decrease inlymphocyte numbers.5. Anti-inflammatory factor suppresses host defenses against infection,probably via an effect on neutrophil emigration.6. Anti-inflammatory factor blocks interactions between cell surfaceCD18 antigens and other molecules.7. Anti-inflammatory factor blocks the adhesion of neutrophils to theendothelium.8. Anti-inflammatory factor promotes the dissociation of adherentneutrophils from the endothelium.9. Anti-inflammatory factor blocks the ability of host lymphocytes torespond to foreign histocompatibility antigens.10. Anti-inflammatory factor blocks the mitogenic response oflymphocytes.

The experimental data obtained in these studies demonstrate dearly thatmilk anti-inflammatory factor has a marked effect on both neutrophilsand lymphocytes. The effects observed may be the result of a directeffect of anti-inflammatory factor on cells per se, or the result of thesuppression (or stimulation) of some other cellular or soluble mediatorwhich indirectly alters the biological activities of cells. It is alsowidely accepted that most pharmacological agents have multiple actionsand it is possible that the anti-inflammatory factor will be found toaffect a number of other, as yet unidentified, biological processes.

EXAMPLE 24 Method of Obtaining Highly Purified MAIF

The isolation of a bioactive factor in a highly purified form suitablefor compositional and structural analysis requires the availability of alarge source of starting material, the development of large-scaleproduction and processing capabilities and the establishment of thenecessary biological and biochemical assays required to confirmbiological activity at every step of the purification procedure. Methodsfor preparation of MAIF from skimmed milk similar to those describedearlier in this application were used to produce a MAIF preparation witha degree of purity suitable for preliminary analytical studies. Apreferred method for the preparation of MAIF in large quantitiessuitable for handling, transportation, storage and processing isdisclosed below. The following procedures will permit the production ofa highly purified form of MAIF in quantities sufficient for compositionand structural studies to be performed.

Materials

Chemicals were all reagent grade. Water used for large-scale preparativeprocedures was sterile water for injection or was freshly preparedpyrogen-free deionized water. Spray-dried rennet whey produced fromhyperimmune milk was used for the production of large-scale amounts ofMAIF.

Standardized MAIF Preparation

In order to compare the MAIF activities in different batches of milkfrom hyperimmune cows or from control cows, a standardized procedureinvolving ultrafiltration and ion exchange chromatography was adopted toprepare a partially purified, highly active sample. A convenient volume(3 to 100 liters) of fresh, cold skimmed milk was subjected toultrafiltration through the Minisete unit (Omega membrane) at 30 psiuntil the volume of filtrate collected was equal to ⅔ of the startingvolume of milk. A convenient volume of standardized, <10,000 daltons(<10K daltons) retentate (250 ml, 500 ml. etc.) was applied to a DEAEcolumn that was 26.7% of the retentate volume. The column was washedwith a volume of deionized water 2 times that of the retentate andeluted with a volume of 0.15 M NaCL equal to 58.3% of the retentatevolume. The MAT activity in standardized DEAE preparations wasdetermined in the neutrophil migration inhibition assay (see below) andwas defined as the percent inhibition of neutrophil migration induced bya 3 mg/120-150 gm rat dose.

Neutrophil migration inhibition activities of samples resulting fromfurther purification of MAIF were compared to the activities ofstandardized DEAE MAIF preparations. All samples were also evaluated fortheir pro-inflammatory potential in a tumor necrosis factor inductionassay (see below).

Highly Purified MAIF Preparation

Ultrafiltration. Ninety liters of fresh, cold (4° C.), non-pasteurizedskimmed milk from hyperimmunized cows was pumped through a 25° C.in-line warming bath and then passed through a Minisette (Filtron)ultrafiltration cassette containing an Omega (model #OS010C01) membranewith a molecular weight cut-off of <10K daltons. Inlet pressure wasmaintained at 30 psi. The resulting retentate (<10K retentate) wascollected on ice until a volume of sixty liters was accumulated. The<10K retentate was used immediately for further purification by ionexchange chromatography or was frozen and stored at 20° C. or waslyophilized.

For large-scale production of MAIF, 18 kg of whey powder fromhyperimmune milk was dissolved in 300 liters of cold (4° C.)pyrogen-free, water. The cold whey was passed through a process filterand was then pumped through a 400 ft² spiral-wound 1000 MW cut-offultrafiltration membrane (DESAL model GE2540F) at a pressure of 300 psiuntil the retentate became ⅓ of the starting volume. Duringultrafiltration the retentate was chilled by recirculation through a 10°C. in-line heat exchanger. The <1K filtrate was collected at 4° C. insterile containers and was frozen at −20° C. and lyophilized.

Ion Exchange Chromatography. Sixty liters of cold fresh <10K retentatewas applied directly to a 37 cm×1.5 cm column (Pharmacia, Model KS370/15) containing 16 liters of diethyl-aminoethyl (DEAE)-Sepharose FastFlow ion exchange resin (Pharmacia #17-0709-05) at a flow rate of 4.8liters/hr. using a low pressure peristaltic pump. The column effluentwas monitored at 280 and 254 nm with a Pharmacia Dual Path UV monitor(Model 19-24217-01). The loaded column was washed with 120 liters ofsterile deionized water to elute unbound material until the 280 nmabsorbance of the effluent returned to baseline. Bound components wereeluted with 35 liters of 0.15 M NaCl. The column eluate was lyophilizedand stored in amber glass vials in the dark. MAIF activity in columneluates was determined in the neutrophil migration inhibition assay (seebelow).

Size Exclusion Chromatography. Partially purified DEAE preparations ofMAIF were separated on a preparative TSK G2500PW (21.5×600 mm) sizeexclusion HPLC chromatography column (TosoHaas, Montgomeryville, Pa.)equilibrated with 0.15 M NH₄ OAc on a Hewlett-Packard Model 1090 HPLCwith an automatic injector and a diode array detector. LyophilizedDEAE-MAIF (100 mg) was dissolved in 0.25 ml of 0.15 M NH₄ OAc andinjected onto the column. The column was eluted with 0.15 M NH₄ OAc at aflow-rate of 5 mi/min. The effluent was monitored at 220 and 280 nm,however, diode array data was stored for all wavelengths between 190 and600 nm. Fractions (9 ml) were collected on an LKB ULTRARAC fractioncollector. To prepare large quantities of MAIF, multiple samples of 100mg each were separated on the TSK column and collected into the same setof tubes. Tubes containing the active factor were pooled, lyophilizedand weighed.

Organic Partition Extraction. MAIF was selectively isolated from boundsalts by an organic partition extraction method in which thesemipurified TSK-MAIF sample was dissolved in distilled water,alkalized, extracted with n-hexane to remove neutral lipids, acidifiedand then extracted with ethyl acetate. 50×100 mg of TSK-MAIF wasdissolved in 2 ml deionized water in a tared extraction vessel. The pHof the solution was adjusted to 8.0 with 4 drops of 0.02 N NH₄ OH. Twomilliliters of n-hexane were added and the mixture was shakenvigorously. The upper phase consisting of hexane was removed, dried andweighted. The remaining aqueous solution was acidified to pH 3.5 with100 drops of citric acid. Five milliliters of ethyl acetate was addedand the mixture was shaken vigorously and centrifuged at 1000 rpm toseparate layers. The ethyl acetate layer was transferred to a taredvessel. The extraction was repeated with a second 5 ml volume of ethylacetate. The ethyl acetate layers were combined, dried under nitrogenand weighed.

Reversed-Phase HPLC. A Zorbax SB-C₁₈ reversed phase HPLC column(Mac-Mod) was used to separate the weakly ionic negatively charged MAIFcomponents freed of hound salts by the ethyl acetate extractionprocedure. Separations were achieved with an ion-pairing isocraticmobile phase consisting of 35% methanol, 20 mM oetylsulfonate, and0.025% trifluoroacetic acid (TFA). Samples of up to 50 mg of extractedmaterial were dissolved in 35% methanol, 0.025% TFA and applied to thecolumn. The column was developed at a flow-rate of 1 ml/min. The columneluate was monitored at 214 and 280 nm. Selected fractions were testedfor MAIF activity in the neutrophil migration inhibition assay (seebelow).

Neutrophil Migration Inhibition Assay. MAIF anti-inflammatory activitywas determined in a pleural neutrophil migration inhibition assay.Female, white laboratory rats weighing 120 to 150 g were injectedintrapleurally with 1.0 ml of 1% kappa carrageenin in PBS to induceneutrophil migration into the pleural cavity. Immediately, the rats wereinjected intraperitoneally with 0.5 ml doses of MALE samples in PBS. PBSwas used as a control. After four hours, the rats were sacrificed,pleural exudates were collected, and the emigrated neutrophils werecounted.

Tumor Necrosis Factor Assay. The capacity of MAIF samples to inducepro-inflammatory cytokines as an indicator of endotoxin contaminationwas evaluated in a tumor necrosis factor induction assay. Test samplesare diluted in RPMI medium and incubated with J774 mouse macrophagecells in a 24-well plate at 37° C. for four hours. The quantity of TNFreleased from the macrophage cells as a result of incubation with testsamples is quantitated by specific TNF ELISA assay. Aliquots of cellsupernatants are incubated in anti-T & monoclonal antibody coated platesovernight at room temperature. Bound TNF is detected in the wells with abiotinylated second antibody developed with a streptavidinperoxidase-TMB substrate system. Developed color is quantitated at 450nm and compared to a standard curve of TNF.

Results

Standardization of Preparative Methods

Reproducible methods involving ultrafiltration and DEAE columnprocedures and analysis of comparable samples in the rat neutrophilmigration inhibition assay were established for the production of highlyactive MAIF preparations. The adoption of standardized purificationmethods permitted quantitation of MAIF activity in specific milkproducts and in large-scale production batches. To evaluate thestandardized method, MAIF was prepared from hyperimmune milk, fromcontrol milk obtained from non-immunized cows and from a commercialpowdered milk and was tested blind in the neutrophil migrationinhibition assay. MAIF samples were tested at doses of 0.5, 1.5, 3, 5,and 8 mg/120-150 gm rats for their ability to inhibit the migration ofneutrophils to inflammatory sites (FIG. 39). MAIF from hyperimmune milkproduced a maximum neutrophil inhibition of more than 70% at the 3 mgdose. Fresh control milk and commercial control milk each produced only18% inhibition at the 3 mg dose. The activity of standardized MAIFprepared from hyperimmune milk was compared with sialic acid and oroticacid, known components of milk believed to have anti-inflammatoryactivity (FIG. 40). Neither the sialic acid nor the orotic acidexhibited any inhibitory activity on the migration of rat neutrophils inthe doses tested.

MAIF Purification by Preparative HPLC

The use of DEAE ion exchange chromatography to prepare MAIF resulted ina 25-fold purification of the anti-inflammatory activity with respect tothe <10K retentate. Analysis of the composition of the DEAE-derived MAIFby size exclusion HPLC (FIG. 41) indicated the presence of two majorcomponents at 17 and 25 minutes and six or more minor components whicheluded between 30 and 60 minutes. Analysis of pooled fractions in theneutrophil migration inhibition assay demonstrated that the majorconcentration of MAIF activity occurred in the 25 minute peak. Theseresults indicated that a preparative size separation step would permitthe preparation of a more highly purified preparation of MAIF. Attemptsto achieve comparable results on a preparative liquid chromatographymedium did not provide adequate separations. A preparative HPLCTSK2500PW column, eluted with 0.15 NM₄ OAc, provided excellentseparation of the 25 minute MAIF peak. DEAE-MAIF was separated on theHPLC column in 100 mg runs and the pooled 25 minute peak waslyophilized. The 0.15 M NH4 OAc elution buffer was selected because allbuffer salts would be completely removable from isolated MAIF samplesduring lyophilization. However, when repeated 100 mg runs of DEAE-MAIFwere fractionated by preparative HPLC, recovered weights of the 25minute peak exhibited only a 10% reduction in weight. Elemental analysisof the MAIF peak indicated that more than 40% of the weight could beattributed to salt. Several salts, including sodium chloride, sodiumacetate and magnesium chloride, were tested to determine their elutionposition on the TSK 2500PW column. Each salt eluted between 35 and 40minutes. This result indicated that salts were specifically hound by theMAIF compound eluting at 25 minutes.

Elimination of Contaminating Salt

In order to free the MAIF compound from bound salts, an organicpartition extraction method was used in which the HPLC purified MAIFpreparation was alkalized to expose negatively charged groups andextracted with hexane to remove hexane soluble contaminants. The aqueousphase was then acidified to protonate negative charges, extracted intoethyl acetate and weighed. The extracted residue exhibited a reductionin weight of more than 96%. Analysis of the dried ethyl acetate fractionin the neutrophil migration inhibition assay demonstrated strong MAIFactivity and required as much as a 10,000 fold reduced dose of thehighly purified MAIF to obtain the same level of neutrophil migrationinhibition as MAIF obtained prior to the organic partition extraction(FIG. 42).

Production of MAIF by <1K Ultrafiltration

A large-scale isolation and purification procedure for the production ofquantities of MAIF sufficient for biochemical characterization,compositional analysis and structural characterization has beendeveloped using powdered hyperimmune whey as the starting material. Wheyis subjected to ultrafiltration on a 1000 MW cut-off ultrafiltrationmembrane. The collected filtrate can be lyophilized or concentrated byreverse osmosis and spray dried. Hyperimmune whey and preparations of<1K filtrate from hyperimmune whey routinely inhibited neutrophilmigration, while <1K filtrate prepared identically from control whey hadlow inhibitory activity (FIG. 43). Since endotoxin, certain salts, and alarge portion of the whey lactose are rejected by the UF1000 membrane,<1K filtrates contain relatively low salt and lactose and are TNFnegative. Organic extracts of the <1K filtrate had activity in theneutrophil migration assay. Analysis of extracts of <1K filtrates onreversed-phase HPLC exhibited a prominent peak at approximately 12minutes (Peak 12) as well as several other components (FIG. 44).Reversed-phase analysis of the organic extracts of hyperimmune wheyrevealed the presence of an identical peak at 12 minutes. Preparationsfrom control milk contained small or undetectable amounts of Peak 12.These results indicate that partition extraction of the <1K filtrateresulted in the removal of significant amounts of salt and othercontaminants and permits the preparation of a more highly purifiedpreparation of MAIF than previously obtainable.

MAIF Purification by Reversed-Phase HPLC

Peak 12 was isolated in preparative quantities by collecting fractionsfrom repeated separations of organic extracts of <1K filtrate onreversed-phase HPLC. Fractions containing Peak 12 were pooled,lyophilized and rechromatographed on reversed-phase HPLC in a mobilephase containing only methanol and water (FIG. 45). Fractions containingPeak 12 were collected and dried. When tested in the neutrophilmigration assay, Peak 12 showed 60% to 80% inhibitory activity at dosesof 3 to 30 nanograms (FIG. 46). When compared together in the neutrophilassay with aspirin, indomethacin and dexamethasone (FIG. 47), Peak 12exhibited 100,000 times the activity of the three anti-inflammatorydrugs all of which were active in the high microgram to milligram range.

These results indicate that a highly purified form of MAIF can beproduced by preparative reversed-phase HPLC of a direct organicextraction of a <1K filtrate obtained from hyperimmune whey. MAIFproduced by this method exhibits high activity when compared to MAIFfrom control milk or to postulated or known anti-inflammatory drugs.MAIF produced by this method is more purified and more highly activethan preparations obtained by previous methods. The use of this novelprocedure will provide sufficient quantities of highly purified materialnecessary for the compositional and molecular structural studies ofMAIF.

All references cited herein are fully incorporated by reference intothis disclosure.

Having now generally described this invention, it will become readilyapparent to those skilled in the art that many changes and modificationscan be made thereto without affecting the spirit or scope thereof. Suchchanges and modifications are also considered aspects of the invention.

In an alternative embodiment, the present invention is directed to amethod for obtaining an anti-inflammatory factor from skimmed milkcomprising the steps of: (i) ultrafiltering the skimmed milk through afilter with a molecular weight cut-off of 1,000 daltons; (ii) collectingthe between 1,000 and 10,000 dalton retentate from step (i); (iii)extracting the retentate from step (ii) by organic partition extractionand obtaining the aqueous extract from the extraction; (iv) separatingthe aqueous extract from step (iii) by reversed-phase HPLCchromatography; and (v) collecting the eluate. This embodiment isparticularly appropriate for large-scale preparation of milkanti-inflammatory factor in quantities suitable for handling,transportation, storage, and processing.

In an embodiment, combining the collected between 1,000 and 10,000dalton retentate with whey protein concentrate or isolate, such as thatdepicted in FIGS. 49-51. Whey protein can be further combined withretentate in FIG. 52 similar to the addition of whey protein in thevarious embodiments shown in FIG. 49.

In an alternative embodiment, the present invention is directed to amethod for obtaining an anti-inflammatory factor from skimmed milkcomprising the steps of: (i) ultrafiltering the skimmed milk through afilter with a molecular weight cut-off of 1,000 daltons; (ii) collectingthe between 5,000 and 10,000 dalton retentate from step (i); (iii)extracting the retentate from step (ii) by organic partition extractionand obtaining the aqueous extract from the extraction; (iv) separatingthe aqueous extract from step (iii) by reversed-phase HPLCchromatography; and (v) collecting the eluate. This embodiment isparticularly appropriate for large-scale preparation of milkanti-inflammatory factor in quantities suitable for handling,transportation, storage, and processing.

FIGS. 48-52 depict various methods for the method for obtaining ananti-inflammatory factor. The method may include admixing or otherwisecombining whey protein with the milk prior to filtration or with theretentate following ultrafiltration. The whey protein may include butnot limited to whey protein concentrate and isolate. The method includespackaging the MPC admixed with whey protein.

FIG. 48 depicts, through a flow chart, a method of obtaining ananti-inflammatory factor that admixes whey protein followingpasteurization and prior to ultrafiltration. At block 4802, raw milk isdelivered or otherwise received. At block 4804, the raw milk that isdelivered or otherwise received is stored for pasteurizing. At block4806, the stored raw milk is pasteurized via a pasteurizer. At block4808, a separator separates for outputting cream at block 4826. At block4824, the separated material is cooled. At block 4826, cream is output.At block 4810, skim milk is output from either of the pasteurizer (Block4806) and/or the separator (Block 4808) and the skim milk is deliveredto the ultrafiltration plant. At block 4812, the skim milk is receivedinto the ultrafiltration plant for ultrafiltration. The skim milk havingadmixed whey protein is ultrafiltered through a filter with a molecularweight cut-off of 1,000 daltons. At block 4814, diafiltration isoptionally applied to remove any additional materials, such asadditional lactose and other soluble minerals. At block 4816, collectedretentate is extracted and cooled via a cooler. At block 4818, theretentate is either output as a liquid or otherwise received at anevaporator/dryer (See FIG. 51).

FIG. 49 depicts, through a flow chart, a method of obtaining ananti-inflammatory factor that admixes whey protein followingpasteurization and prior to ultrafiltration. At block 4902, raw milk isdelivered or otherwise received. At block 4904, the raw milk that isdelivered or otherwise received is stored for pasteurizing. At block4906, the stored raw milk is pasteurized via a pasteurizer. At block4908, a separator separates for outputting cream at block 4926. At block4924, the separated material is cooled. At block 4926, cream is output.At block 4910, skim milk is output from the pasteurizer. At block 4912,whey protein is added to the skim milk. At block 4914, the skim milkhaving admixed whey protein is received into the ultrafiltration plantfor ultrafiltration. The skim milk having admixed whey protein isultrafiltered through a filter with a molecular weight cut-off of 1,000daltons. At block 4916, diafiltration is optionally applied for removingadditional lactose and other soluble materials. At block 4918, collectedretentate is extracted and cooled via a cooler. At block 4920, theretentate is either output as a liquid or otherwise received at anevaporator/dryer.

FIG. 50 depicts, through a flow chart, a method of obtaining ananti-inflammatory factor that admixes whey protein with retentatefollowing ultrafiltration. At block 5002, raw milk is delivered orotherwise received. At block 5004, the raw milk that is delivered orotherwise received is stored for pasteurizing. At block 5006, the storedraw milk is pasteurized via a pasteurizer. At block 5008, a separatorseparates for outputting cream at block 5026. At block 5024, theseparated material is cooled. At block 5026, cream is output. At block5010, skim milk is output from the pasteurizer. At block 5012, the skimmilk is received into the ultrafiltration plant for ultrafiltration. Atblock 5014, the skim milk is ultrafiltered through a filter with amolecular weight cut-off of 1,000 daltons. At block 5016, the retentateis extracted from the ultrafiltration process and whey protein iscombined with the retentate following the extraction or otherwiseadmixed with the retentate subsequent to ultrafiltration. At block 5018,the extracted retentate is cooled via a cooler. At block 5020, the MPCis either output as a liquid or otherwise received at anevaporator/dryer.

FIG. 51 depicts, through a flow chart, a method of obtaining ananti-inflammatory factor that produces MPC powder of retentate and/orwhey protein concentrate or isolate. At block 5102, retentate from theultrafiltration plant is received. At block 5104, the received retentatewith whey protein is provided to an evaporator and evaporated. At block5106, the retentate is received by a spray dryer following evaporationby the evaporator and under goes spay-drying as a step in creating MPC.At block 5108, a bagging machine receives MPC that was spray dried bythe spray dryer. The bagging machine bags the MPC created bagged MPC. Atblock 5110, the bagged MPC is palletized. In one embodiment, wheyprotein may be admixed during or between any of the blocks identifiedherein. For example, whey protein may be added to the MPC at the timethe MPC is bagged.

What is claimed is:
 1. A method for purifying an anti-inflammatoryfactor from skimmed milk comprising: (i) ultrafiltering said skimmedmilk through a filter with a molecular weight cut-off of 1,000 daltons;(ii) collecting the >1,000 dalton retentate from step (i); (iii)extracting the retentate from step (ii) by organic partition extractionand obtaining the organic extract from said extraction; (iv) separatingthe organic extract from step (iii) by reversed-phase HPLCchromatography; and (v) collecting the eluate.
 2. The method of claim 1,further comprising admixing whey protein with the >1000 daltonsretentate collected from step (ii).
 3. The method of claim 1 whereinsaid skimmed milk is hyperimmune skimmed milk.
 4. The method of claim 3wherein said hyperimmune skimmed milk is whey.
 5. The method of claim 1,wherein said organic partition extraction comprises: (i) extracting withhexane and NH4 OH (ii) reextracting the aqueous phase from step (i) withethyl acetate; and (iii) collecting said ethyl acetate extract.
 6. Themethod of claim 5, wherein said reversed-phase HPLC chromatographycolumn is a Zorbax SB-C18 column.
 7. An anti-inflammatory factor inhighly purified form produced by a process comprising: (i)ultrafiltering skimmed milk through a filter with a molecular weightcut-off of 1,000 daltons; (ii) collecting the >1,000 dalton retentatefrom step (i); (iii) extracting the retentate from step (ii) by organicpartition extraction and obtaining the organic extract from saidextraction; (iv) separating the organic extract from step (iii) byreversed-phase HPLC chromatography; and (v) collecting the eluate. 8.The method of claim 7, further comprising admixing whey protein withthe >1000 daltons retentate collected from step (ii).
 9. Theanti-inflammatory factor of claim 7 wherein said skimmed milk ishyperimmune skimmed milk.
 10. The anti-inflammatory factor of claim 9wherein said hyperimmune skimmed milk is whey.
 11. The anti-inflammatoryfactor of claim 1 wherein said organic partition extraction furthercomprises: (i) extracting with hexane and NH4 OH; (ii) reextracting theaqueous phase from step (i) with ethyl acetate; and (iii) collectingsaid ethyl acetate extract.
 12. An anti-inflammatory factor insubstantially or highly purified form produced by the method of claim 1.13. A method for inhibiting neutrophil migration comprisingadministering to an animal the milk anti-inflammatory factor obtained bythe method of claim 1 at a dose sufficient to inhibit said neutrophilmigration.
 14. A method for inhibiting the inflammatory responsecomprising administering to an animal the milk anti-inflammatory factorobtained by the method of claim 1 at a dose sufficient to inhibit saidinflammatory response.
 15. A method for inhibiting neutrophils fromadhering to endothelial cells in a mammal, wherein said method comprisesadministering to said mammal the milk anti-inflammatory factor obtainedby the method of claim 1 at a dose sufficient to inhibit said adherence.16. A method for detaching neutrophils which have adhered to endothelialcells in a mammal, wherein said method comprises administering to saidmammal the milk anti-inflammatory factor obtained by the method of claim1 at a dose sufficient to cause said detachment.
 17. The method of claim16, wherein said neutrophils have adhered to said endothelial cells inresponse to platelet activating factor.
 18. A method for inhibiting theinteraction between CD18 cell-surface antigens present in a mammal andmolecules which are capable of binding to said antigens, wherein saidmethod comprises administering to said mammal the milk anti-inflammatoryfactor obtained by the method of claim 1 at a dose sufficient to inhibitsaid interaction.
 19. A method for inhibiting the emigration ofleukocytes from the venous system of a mammal, wherein said methodcomprises administering to said mammal the milk anti-inflammatory factorobtained by the method of claim 1 at a dose sufficient to inhibit saidemigration.
 20. A method for suppressing the mitogenic response oflymphocytes in a host mammal to foreign antigens, wherein said methodcomprises administering to said mammal the milk anti-inflammatory factorobtained by the method of claim 1 at a dose sufficient to suppress saidmitogenic response.